Mycobacterial recombinants and peptides

ABSTRACT

Recombinant 540 amino acid residue and 517 amino acid residue proteins encoded by the genome of Mycobacterium tuberculosis are disclosed as are vectors for propagating their DNA sequences and expressing the proteins. Also disclosed are methods for using those proteins. Peptides that correspond substantially to the sequences of those proteins and methods of their use are also disclosed, as are polymers containing peptide repeating units corresponding to the 540 residue protein and also polymers containing 517 protein pentapeptides as repeating units.

CROSS REFERENCE TO A RELATED APPLICATION

This is a continuation-in-part of co-pending U.S. patent applicationSer. No. 019,529 filed on Feb. 26, 1987, that is incorporated herein byreference.

Description

1. Technical Field

The present invention relates to recombinant proteins and peptidesrelated to mycobacteria, and particularly to proteins of Mycobacteriumtuberculosis that are coded for by adjacent open reading frames oncomplementary DNA strands of the genome and vectors for propagating andexpressing those recombinants, as well as to peptides that correspondsubstantially in sequence to portions of those proteins.

2. Background Art

The mycobacteria are a diverse collection of acid-fast, gram-positivebacteria some of which cause important human and animal diseases[reviewed in Bloom et al., (1983), Rev. Infect. Dis., 5:765-780; andChaparas, (1982), CRC Reviews in Microbiology, 9:139-1971. In man, thetwo most common mycobacteria-caused diseases are tuberculosis andleprosy, which result from infections with Mycobacterium tuberculosisand Mycobacterium leprae, respectively. These two diseases afflict morethan 65 million individuals world-wide and result in over 4 milliondeaths annually, Bloom et al., (1983), Rev. Infect. Dis., 5:765-780.

The pathogenicity of these mycobacterial infections is closely tied tothe host's immune response to the invading mycobacterium [Chaparas,(1982), CRC Reviews in Microbiology, 9:139-197; Collins, (1982), Am.Rev. Respir Dis , 125:42-49; Dannenberg, (1982), Am. Rev. Respir. Dis.,125:25-29; and Grange, (1984), Adv. Tuberc. Res., 21:1-78]. Not onlydoes M. tuberculosis infect and grow within cells of the host's immunesystem, primarily the aveolar macrophage, but also it is the host'scellular immune response that plays the key roles in immunity frominfection, containment of the infection at the initial focus ofinfection, progression or regression of the infection, and tissue damageor destruction at the foci of infection [Chaparas, (1982), CRC ReviewsIn Microbiology, 9:139-197; Collins, (1982), Am. Rev. Respir. Dis.,125:42-49; Dannenberg, (1982),Am. Rev. Respir. Dis., 125:25-29; andGrange, (1984), Adv. Tuberc. Res., 21:1-78]. In addition, the standardmethod of detecting an M. tuberculosis infection, the tuberculin skintest, actually measures the host's cellular immune response to themycobacterium [Snider, (1982), Am. Rev. Respir. Dis., 125:108-118]. Themycobacterial components that are important in eliciting the cellularimmune response are not yet well defined.

A number of studies have attempted to define the mycobacterial antigensby standard biochemical and immunological techniques including theanalysis of the target antigens of monoclonal hybridoma antibodiesdirected against mycobacteria [Daniel et al., (1978), Microbiol. Rev.,42:84-113; Engers et al., (1985), Infect. Immun., 48:603-605; Engers etal., (1986), Infect. Immun., 51:718-720; Grange, (1984), Adv. Tuberc.Res., 21:1-78; Ivanyi et al., (1985), Monoclonal Antibodies AgainstBacteria (A. J. L. and E. C. Macario, eds.) Academic Press, Inc. NewYork. pp. 59-90; and Stanford, (1983), The Biology of the Mycobacteria(Ratledge and Stanford, eds.), Academic Press, London, vol. 2, pp.85-127].

One particular antigen, a 65 kilodalton (KD) protein, is present in awide range of mycobacterial species and has been most intensivelystudied as an antigen of M. leprae [Emmrich et al., (1986), J. Exp.Med., 163:1024-1029; Gillis et al., (1985), Infect. Immun., 49:371-377;Young et al., (1985), Nature, 316:450-452; and Mehra et al., (1986)Proc. Natl. Acad. Sci. USA, 83:7013-7017]. This antigen has beendesignated the 65KD antigen or the cell wall protein-a (CWP-a) antigensince it appears to a co-purify with cell walls in some isolationprocedures [Gillis et al., (1985), Infect. Immun., 49:371-377].

In Western blot assays, monoclonal antibodies directed against thisantigen react with two major components in an M. leprae extract thatmigrate with apparent sizes of 55,000 and 65,000 daltons, and reactoccasionally with smaller components as well [Engers et al., (1985),Infect. Immun., 48:603-605 and Gillis et al., (1985), Infect. Immun.,37:172-178]. It is not known if these species represent discreteproteins or precursors and products, or result from chemical orenzymatic cleavage during isolation. In other species, such as M.gordonae, only a single species of about 65,000 daltons is detected withthe monoclonal antibodies [Gillis et al., (1985), Infect. Immun.,49:371-377].

The 65KD antigen is one of the major immunoreactive proteins of themycobacteria. This antigen contains epitopes that are unique to a givenmycobacterial species as well as epitopes that are shared amongstvarious species of mycobacteria [Engers et al., (1985 ), Infect. Immun.,48:603-605 and Gillis et al., (1985), Infect. Immun., 49:371-377]. Inaddition, some other antigens that appear to be expressed by only onemycobacterial species are also found to contain epitopes expressed inother mycobacterial species. [Kingston et al., (1987) Infect. Immun.,55:3149.]

As discussed hereinafter, it is now found that purified 65KD antigen canelicit a strong delayed-type hypersensitivity reaction in experimentalmammals infected with M. tuberculosis. Antibodies directed against thisprotein can also be detected in the sera of patients with tuberculosisor leprosy, and T-cells reactive with this antigen can be isolated frompatients with leprosy or tuberculosis as well as from BCG-vaccinatedpersons [Emmrich et al., (1986), J. Exp. Med., 163:1024-1029; Engers etal., (1986), Infect. Immun., 51:718-720; Mustafa et al., (1986), Nature,319:63-66; and Thole et al., (1985), Infect. Immun., 50:800-806].Overall, the 65KD antigen appears to be a major, medically important B-and T-cell immunogen and antigen in humans.

BRIEF SUMMARY OF THE INVENTION

The present invention relates to DNA sequences, vectors containing theDNA sequences, proteins, recombinant proteins, peptides, their method ofmanufacture and use that relate to a Mycobacterium tuberculosis. Moreparticularly, those DNA sequences, vectors, proteins, recombinants andpeptides relate to two proteins denominated the 540 (65KD) and 517proteins that are coded for by adjacent open reading frames oncomplementary DNA strands of the mycobacterial genome The peptidescorrespond substantially to portions of those proteins.

One embodiment of the invention contemplates an isolated DNA moleculethat consists essentially of a nucleotide sequence, from right to leftand in the direction from 5'-end to 3'-end, corresponding to thesequence represented by the formula of FIG. 2 from about position 3950to about position 2390 and in a consistent reading frame coding for a517 amino acid residue protein of Mycobacterium tuberculosis. Morepreferably, that sequence extends from position 3948 through position2398.

A plasmid vector that comprises a replicon operationally linked to aforeign DNA sequence such as that above and that is capable ofreplicating that foreign DNA sequence in a replication/expression mediumis also contemplated herein, particularly where thereplication/expression medium is a unicellular organism, such as abacterium like E coli. The plasmid vector typically includessequence-encoded signals for initiation and termination of transcriptionthat are operationally linked to the foreign DNA sequence and arecompatible with the replication/expression medium for transcribing aproduct coded for by the foreign DNA sequence. Further, it can include atranslation initiation codon and a translation termination codon, eachof which is operationally linked to the 5'-end and the 3'-end,respectively, of the DNA sequence, and are compatible with thereplication/expression medium for expressing a protein product coded forby the foreign DNA sequence

Still further, the 5'-end of the foreign DNA sequence can beoperationally linked in translational reading frame to the 3'-end of asecond DNA sequence that codes for a second protein or protein fragmentor portion, such as the beta-galactosidase molecule The protein productexpressed by that vector is thus a fusion protein that contains thesecond protein or protein fragment or portion at the amino-terminus andthe first-named protein at the carboxy-terminus of the fusion protein;i.e., the fragment or portion of the second protein is at theamino-terminus of the first-named protein.

A culture comprising bacteria that contain a previously describedplasmid vector in an aqueous medium appropriate for the expression ofthe 517 amino acid residue protein of M. tuberculosis is alsocontemplated.

The present invention further contemplates a method for producing a 517amino acid residue protein of M. tuberculosis. That method comprises thesteps of culturing a replication/expression medium containing a plasmidvector for replicating and expressing foreign DNA sequence containedtherein. That vector contains a foreign DNA sequence that correspondssubstantially to the previously mentioned DNA molecule that encodes thesequence of the 517 M. tuberculosis protein. The vector also containsoperatively linked nucleotide sequences regulating replication andexpression of the foreign DNA sequence. The culturing is carried outunder conditions suitable for expression of the protein that is encodedby the foreign DNA. The expressed protein encoded by that foreign DNAsequence is thereafter harvested. Culture is typically carried out usingunicellular organisms as the replication/expression medium. Suchunicellular organism are typically bacteria as described previously.

A method for determining previous immunological exposure of a mammalianhost to Mycobacterium tuberculosis or Mycobacterium bovis is alsocontemplated. This method comprises the following steps. An inoculumthat consists essentially of the purified 65KD (540) protein or animmunologically active portion thereof coded for by the DNA sequence ofFIG. 2 is administered intradermally to an assayed mammalian host. Thatprotein is dissolved or dispersed in a physiologically tolerable diluentand is present in that diluent in an amount effective to induce erythemaand induration in a mammalian host previously immunized with M.tuberculosis or M. bovis. The mammal is maintained for a time period ofabout 24 to about 72 hours, and thereafter is assayed for the presenceof erythema and induration at the site of the intradermal administrationat the end of that time period. In one aspect of this method thepurified 65KD protein is obtained from a mycobacterium such as M.tuberculosis. In another aspect of this method, the purified protein isa recombinant 65KD protein, or a recombinant fusion protein thatcontains a portion of a beta-galactosidase molecule peptide-bonded tothe amino-terminus of the 65KD protein, or to the amino-terminus of animmunologically active portion thereof. This type of assay is usuallyreferred to as a delayed cutaneous hypersensitivity (DCH) assay.

Still another aspect of the invention contemplates an inoculum thatconsists essentially of the purified 65KD (540 amino acid residue)protein antigen or a fusion protein that is coded for by the sequence ofFIG. 2. That protein antigen is dissolved or dispensed in aphysiologically tolerable diluent, and is present in the diluent in anamount that is effective to induce erythema and induration a mammalianhost previously immunized with M. tuberculosis or M. bovis. The 65KDprotein antigen of the inoculum can be one of the proteins useful in themethod described immediately above.

Still a further aspect of the invention is a peptide that consistsessentially of a 5 to about 40 amino acid residue sequence thatcorresponds substantially to a sequence of the 540 amino acid residueprotein or the 517 amino acid residue protein coded for by the DNAprotein sequence of FIG. 2. More preferably, the peptide contains about10 to about 20 amino acid residues

Preferred peptides include those having a sequence, written from left toright in the direction from amino-terminus to carboxy-terminus usingsingle letter symbols, that corresponds to a formula selected from thegroup consisting of

    ______________________________________                                        A V L E D P Y I L L V S S K V                                                                        (22; 211-225);                                         L L V S S K V S T V K D L L P                                                                        (23; 219-233);                                         L L P L L E K V I G A G K P L                                                                        (24; 231-245);                                         A I L T G G Q V I S E E V G L                                                                        (30; 291-305);                                         I A F N S G L E P G V V A E K                                                                        (46; 451-465);                                         A R R G L E R G L N A L A D A V K V                                                                  (58; 11-28);                                           E K I G A E L V K E V A K K                                                                          (59; 67-78);                                           G L K R G I E K A V E K V T E T L                                                                    (60; 114-130); and                                     I E D A V R N A K A A V E E G                                                                        (68; 394-408);                                         ______________________________________                                    

wherein each first parenthesized number refers to the Peptide number ofTables 2 and 4, hereinafter, and the second hyphenated numbers refer tothe position in the sequence of the 540 amino acid residue-containingprotein whose complete amino acid residue sequence and genomic sequenceare illustrated in FIGS. 2A and 2B.

Further contemplated is a method for ascertaining the presence ofmycobacterially-exposed or mycobacterially-immune, i.e., previouslyimmunologically exposed, mononuclear cells such as T cells in a bodysample. Here, mononuclear cells from a mammalian host to be assayed areadmixed and contacted in an aqueous cell culture medium with astimulating amount of both antigen presenting cells and a preferredpeptide antigen to form a stimulatory cell culture. That stimulatorycell culture is maintained for a time period sufficient for immunemononuclear cells present to be stimulated and to evidence theirstimulation. The presence of mononuclear cell stimulation is thereafterdetermined. This assay can be carried out in vivo as a DCH assay wherethe antigen presenting cells are endogenous cells such as macrophagesand the aqueous medium is supplied by the blood and lymph. The assay canalso be carried out in vitro. A polymer having an above peptide asrepeating units can also be used as the antigen.

An assay kit containing a preferred peptide in a container in an amountsufficient to carry out at least one assay as described immediatelyabove is also contemplated.

The invention still further contemplates a vaccine against mycobacteriasuch as M. tuberculosis. The vaccine comprises a physiologicallytolerable diluent containing as immunogen an immunizing effective amountof (i) a peptide antigen containing 5 to about 40 residues, and morepreferably about 10 to about 20 residues, whose amino acid residuesequence corresponds substantially to a sequence of a mycobacterial 65KDprotein and that is capable of stimulating mycobacterially-immune Tcells having a phenotYpe selected from the group consisting of T4⁺ andT8⁺ or (ii) a polymer having said peptide antigen as repeating units.Preferably, the mycobacteria is M. tuberculosis. The mycobacteria towhich the T cells are immune is the same mycobacterial species to whichthe vaccine is directed.

Yet another aspect of the present invention is a polymer that comprisesa plurality of pentapeptide repeating units. Each of those pentapeptiderepeating units consists essentially of a sequence, written from left toright in the direction of amino-terminus to carboxy-terminus,represented by a formula

N N N I G; or

X G N Z G,

wherein X is an amino acid residue selected from the group consisting ofF, S, T, L, D, and I; and Z is an amino acid residue selected from thegroup consisting of T, I, L, S and V. In a further aspect of thisinvention, the pentapeptide repeating units are bonded together bypeptide bonds, whereas in yet another aspect, the pentapeptide repeatingunits are bonded together by oxidized cysteine residues at the terminiiof those repeating units.

BRIEF DESCRIPTION OF THE DRAWINGS

In the drawings forming a portion of this disclosure:

FIG. 1 is a schematic restriction map of recombinants expressing the M.tuberculosis 65KD antigen. The portion of the genome containing the 65KDprotein is shown as the heavy line at the top of the Figure along withthe relative positions (short perpendicular lines abutting the heavyline) of restriction endonuclease cleavage sites. The single lettersadjacent those short lines are indicia of the endonclease that cleavesthe genome at the indicated sites, and are: A=SacI, B=Bgl II, K=KpnI,M=BamHI, P=PstI, R=EcoRI, S=Sal I, V=PvuII, and X=XhoI.

Twenty of the recombinants discussed herein are enumerated along theright-hand margin of the Figure opposite the schematic linerepresentations of the respective genomic portion contained by eachrecombinant. The lengths and positions of those genomic portionsrelative to the genome of the 65KD protein are shown by the relativelengths and positions of the lines. Dashes at the termini of the firstsix shorter lines indicate that those recombinants contained additionalbase pairs, but the source and sequences of those additional base pairsis presently uncertain.

DNA was isolated from phage stocks of the recombinants expressing the65KD antigen as described by Helms et al. (1985) DNA 4:39-49, and arestriction enzyme cleavage site map was constructed.

FIG. 2 shows the nucleotide sequence of the region containing the M.tuberculosis 65KD antigen and 517 protein genes, and is provided as foursheets labeled 2A, 2B, 2C and 2D. The deduced amino acid residuesequences of the two long open reading frames (ORFs) capable of codingfor proteins containing 540 and 517 amino acid residues, respectively,are shown using the one letter code over (540) or under (517) theappropriate triplets. Asterisks above or below the respective sequencesindicate the positions of stop codons (TGA, TAG or TAA) in the DNAsequences. Each sequence is shown as beginning with the first methionine(M) residue in phase with the ORF and downstream of the nearest upstreamstop codon.

FIG. 3 is a schematic representation of the open reading frames found inthe portion of the mycobacterial DNA sequence that codes for the 65KDantigen. The heavy line near the top of the Figure represents a portionof the genome that includes the 540 and 517 proteins. The shorter,arrow-tipped lines beneath the heavy line indicate DNA sequences thatexceed 120 amino acid residues in length. Putative initiation tripletsare identified on the shorter lines by the letter "M" (AUG) or theletter "V" (GUG) at the 5'-end of each open reading frame in therelatively shorter sequences illustrated beneath the heavy line. Arrowsindicate the coding direction.

FIG. 4 is a photograph of a Western blot analysis of products of the 540amino acid residue open reading frame, and contains two panels, A and B.Cells were grown and induced (except for lane 2, Panel A) and crudeextracts were prepared as described in the Materials and Methodssection, hereinafter. For each lane, except lane 5, 200 micrograms (ug)of protein were electrophoresed on a 10% Laemmli gel, and transferred tonitrocellulose. For lane 5, 500 ug of protein were loaded. Theimmobilized proteins were reacted with the IT-13 antibodies andvisualized, as discussed hereinafter.

For Panel A, the proteins in the lanes were: lane 1, JM83; lane 2, JM83(pTB22) uninduced; and lane 3, JM83 (pTB22) induced with IPTG. For PanelB, the proteins in the lanes were: lane 1, JM83 (pTB12); lane 2, Y1089(λSK116); lane 3, Y1089 (λRY3146); lane 4, BNN97 [E. coli C600containing λgt11]; and lane 5, JM83 (pTB12).

DEFINITIONS

The following abbreviations and symbols are used herein.

    ______________________________________                                        bp                                                                                         base pair(s)                                                                  kbp                                                                            1000 bp                                                                      KD                                                                             kilodalton(s)                                                                M.sub.r                                                                        apparent relative molecular mass                                             DNA                                                                            deoxyribonucleic acid                                                        replicon                                                                       the unit that controls                                                         individual acts of replication;                                               it has an origin at which                                                     replication is initiated and it                                               can have a terminus at which                                                  replication stops.                                             ______________________________________                                    

When used in a context describing or depicting nucleotide sequences, thepurine or pyrimidine bases forming the nucleotide sequence are depictedas follows:

    ______________________________________                                                           deoxyadenyl                                                                   G                                                                              deoxyguanyl                                                                  C                                                                              deoxycytosyl                                                                 T                                                                              deoxythymidyl                                             ______________________________________                                    

In describing a nucleotide sequence each three-letter tripletconstituted by the bases identified above represents a trinucleotide ofDNA (a codon) having a 5'-end on the left and a 3'-end on the right ofthe upper sequence of FIG. 2, and a 5'-end on the right and a 3'-end onthe left of the lower, complementary sequence.

The word "antigen" is often used in the art for an entity that is boundby an antibody. The word "immunogen" is often used in the same contextfor the entity that induces the production of antibodies. Where theantigen and immunogen are the same entity, both are often referred to anantigen.

The present invention deals with antigens and immunogens in the abovecontext, which context typically relates to B cells and antibodies.Notwithstanding the B cell/antibody context, the present invention alsocontemplates T cells.

A more general definition of immunogen and antigen apply in the contextof T cells and T cell stimulation. In that more general definition, an"antigen" is an entity acted upon by a component of the immune system,and an "immunogen" is an entity that initiates an immune systemresponse. Where antigen and immunogen are the same, both are referred toas an antigen. An "immunologically active" entity interacts withantibodies or T cells, or can initiate a cellular or humoral immuneresponse.

DETAILED DESCRIPTION OF THE INVENTION I. Overview

In studies discussed hereinafter, the isolation of the gene encoding theM. tuberculosis 65KD antigen and the determination of its nucleotidesequence are reported. The sequence contains an open reading frameencoding 540 amino acid residues or about 60,000 daltons, whichcorresponds to the 65KD antigen. A second long open reading framecapable of encoding a protein of 517 amino acids was also found on themycobacterial DNA fragment containing the 65KD antigen gene, adjacent tothat gene. Interestingly, the central region of the deduced amino acidresidue sequence of the 517 amino acid protein contains several tandemlyarranged, perfect and imperfect repeats of a five amino acid residuesequence. This feature is reminiscient of the features of the sequenceof the major T-cell antigen of the sporozoite stage of the humanmalarial parasite [Nussenzweig et al., (1985), Cell, 42:401-403].

II Results A. Isolation and Analysis or Recombinants

To isolate the gene that encodes the 65KD antigen, monoclonal hybridomaantibodies directed against this antigen were used to screen a proteinexpression library constructed with mycobacterial DNA. An expressionlibrary was chosen since it was not known a priori if the M.tuberculosis genes would 1 in E coli. Such a recombinant DNA library hasbeen constructed by Young et al., (1985),

Proc. Natl. Acad. Sci. USA, 82:2583-2587, and contains genomic DNAfragments of M. tuberculosis inserted into the expression site of thelambda-gt11 (λgt11 ) vector. In this system, the inserted codingsequences can be expressed as a fusion protein with beta-galactosidase.The 65KD antigen-specific monoclonal hybridoma antibodies used in thesestudies were generated in the laboratories of Dr. T. M. Buchanon(Pacific Medical Center, University of Washington, Seattle WA) and Dr.J. Ivanyi (MRC Tuberculosis Unit, Hammersmith Hospital, London) and wereobtained from the Steering Committee on the Immunology of Tuberculosisof the World Health Organization.

As the initial antibody probe, a pool containing three monoclonalantibodies directed against the 65KD antigen was used (IT-13, IT-31, andIT-33). Thirty-eight positive signals were detected in a screen of about8x105 recombinant phage.

The phage corresponding to the positive signals were twice plaquepurified and then assayed for reactivity with the individual antibodies.The results of that purification and assay are shown in Table 1, below.

                  TABLE 1                                                         ______________________________________                                        Patterns of Antibody Reactivities.sup.l                                                  Reactivity With Antibodies                                         Number of Clones                                                                           IT-13       IT-31   IT-33                                        ______________________________________                                        28           +           +       +                                            3            +           +       -                                            3            -           +       +                                            2            -           +       -                                            2            -           -       +                                            ______________________________________                                         .sup.l Recombinant clones expressing antigens reactive with the 65KD          antigen specific monoclonal antibodies IT13, IT31, and IT33 were isolated     as described in the text. For the initial screen, a pool of the three         antibodies that contained a 1:1000 dilution of each antibody was used to      screen a total of about 8 × 10.sup.5 recombinant phage from the         lambda gtllM. tuberculosis library. To determine which monoclonal antibod     reacted with which of the 38 plaquepurified recombinants, about 100           plaqueforming units (pfu) of each recombinant phage were inoculated in        small spots on a lawn of E. coli Y1090. The phage were allowed to grow,       and were induced to synthesize the foreign proteins as described herein.      The filters were then reacted with a 1:1000 dilution of one of the            monoclonal hybridoma antibodies as described in Materials and Methods.   

¹ Recombinant clones expressing antigens reactive with the 65KD antigenspecific monoclonal antibodies IT-13, IT-31, and IT-33 were isolated asdescribed in the text. For the initial screen, a pool of the threeantibodies that contained a 1:1000 dilution of each antibody was used toscreen a total of about 8×10⁵ recombinant phage from the lambda gt11-M.tuberculosis library. To determine which monoclonal antibody reactedwith which of the 38 plaque-purified recombinants, about 100plaque-forming units (pfu) of each recombinant phage were inoculated insmall spots on a lawn of E. coli y1090. The phage were allowed to grow,and were induced to synthesize the foreign proteins as described herein.The filters were then reacted with a 1:1000 dilution of one of themonoclonal hybrodoma antibodies as described in Materials and Methods.

Twenty-eight of the recombinants produced antigens that reacted with allthree antibodies, whereas ten recombinants produced antigens thatreacted with one or two of the antibodies. Overall, the patterns ofreactivity indicate that although the three antibodies react with thesame mycobacterial antigen, each recognizes a different epitope on thatantigen. Richard A. Young (Whitehead Institute, M.I.T.) has alsoscreened this λgt11-M. tuberculosis library with one of these antibodies(IT-13) and detected 10 additional recombinants [Young et al., (1985)Proc. Natl. Acad. Sci. USA, 82:2583-258]. These recombinants were notassayed for reactivity with the other antibodies.

DNA was isolated from twenty of the recombinants expressing the 65KDantigen and a restriction enzyme cleavage site map was deduced for thisregion of the mycobacterial genome (FIG. 1). In most of therecombinants, the mycobacterial DNA insert was flanked by EcoRI sites asexpected from the way in which the library was constructed.

However, in 6 of the 20 recombinants studied, only one of the expectedEcoRI sites was present. This observation raises the possibility that asignificant fraction of the recombinant phage in this library might havearisen from the insertion of a fragment containing only one functionalEcoRI site into the λgt11 EcoRI site or that some clones might haveundergone some sort of recombination, rearrangement or deletion eventduring propagation that removed one of the EcoRI sites.

The deduced restriction map is in good agreement with the published mapof the gene for the M. bovis 65KD antigen [Thole et al., (1985), Infect.Immun., 50:800-806] except for the presence of two additional SMaI sitesin the M. tuberculosis gene. The map does not match well with that ofthe M. leprae 65KD antigen gene [Young et al., (1985), Nature,316:450-452]. This is not unexpected given that based on DNA homologystudies, M. tuberculosis is at least 90% homologous with M. bovis andonly about 30% homologous with M. leprae, Athway et al., (1984), Int. J.Syst. Bacteriol., 34:371-375; Imaeda, (1985) Int. J. Sys. Bacteriol.,35:147-150.

To determine the nucleotide sequence of this region of the mycobacterialgenome, several fragments from the gt11 recombinants were subcloned intothe plasmid vector pUC19. The majority of the sequence of this regionwas determined from a subclone (pTB7) of the 1.4 kilobase pair (kbp)EcoRI fragment of λSK7 and a subclone (pTB9) of the 2.6 kbp EcoRIfragment of λRY3143. The sequence across the EcoRI site at the junctionof these two fragments was determined from a fragment isolated from asubclone (pTB11) of the 2.8 kbp KpnI fragment of λSK119. The sequence ofthe region 5' to the 2.6 kbp EcoRI fragment was determined from asubclone (pTB12) of the 2.4 kbp KpnI fragment of λSK119.

In all, the nucleotide sequence of 4380 base pairs of the mycobacterialDNA was determined by a combination of the Sanger dideoxy chaintermination [Sanger et al., (1980), J. Mol. Biol., 143:161-178] andMaxam-Gilbert chemical degradation [Maxam et al., (1976), Proc. NatlAcad. Sci. USA, 74:560-564] sequencing techniques. The sequence is shownin FIG. 2.

As expected for M. tuberculosis genomic DNA [Wayne et al., (19681, J.Bacteriol., 96:1916-19191, the base composition of this fragment wasabout 66% G+C. The high G+C content increased the chances of sequencingartifacts due to compressions, and made it imperative that the sequenceswere determined for both strands in all regions.

B. Open Reading Frames

The sequence contains five open reading frames (ORFs) that begin with anATG triplet and contain greater than 120 amino acids. Two of theseexceed 200 amino acids in length. One can encode 517 amino acids and theother 540 amino acids.

There are an additional three open reading frames of 140-190 amino acidresidues in length that do not contain an initiation ATG triplet but docontain a GTG triplet. It is not known if a GTG triplet can function asa translation initiation triplet in mycobacteria. The locations of theseeight open reading frames are shown schematically in FIG. 3. No portionsof the deduced amino acid sequences of any of these open reading framesdisplayed any significant homologies with sequences in the ProteinSequence Database of the Protein Identification Resource.

It should be noted that although an open reading frame exceeding 100amino acids would be considered to have a high probability of beingexpressed into protein in most bacteria, this may not be true for themycobacteria. That is, given that the G+C content of the insert is about66%, a translation termination triplet (TAA, TAG or TGA) would beexpected to occur on average about once every 41 amino acids as comparedto about once every 21 amino acids in a genome with a G+C content of50%. Perhaps then, an open reading frame of as many as 150-200 aminoacids might be due to the random distribution of termination tripletsrather than signifying possible biologic importance. As such, only thetwo very long open reading frames that could encode proteins of 517 and540 amino acid residues, respectively, are described herein.

The 540 Amino Acid Residue ORF Corresponds to the 65KD Antigen

One of the long open reading frames begins with an ATG triplet atpositions 252-254 of the DNA sequence and extends to a TGA triplet atpositions 1872-1874. This ORF encodes 540 amino acids. To determine ifthis open reading frame corresponded to the gene for the 65KD antigen,the 1511 bp BamHI-KpnI fragment from pTB12 (residues 438-1948 of thesequence represented in FIG. 2), which contains the majority of thisopen reading frame, was inserted into BamHI-KpnI-cleaved pUC19. In thisconstruct, denominated pTB22, the open reading frame is expressed usingthe lacZ transcription and translation initiation signals present in thepUC19 vector, and results in the production of a fusion proteincontaining 15 amino acid residues at the amino-terminus encoded by thelacZ gene of pUC19 followed by 478 amino acids of the mycobacterial openreading frame.

Crude extracts were prepared from cells containing this plasmid, andwere tested for reactivity with 65KD antigen-specific antibodies inWestern blot analyses. The reactivity with monoclonal antibody IT-13 isshown in panel A of FIG. 4. In all, five different monoclonal antibodiesspecific for the 65KD antigen reacted with a species in the crudeextract that migrated with an apparent relative molecular mass (M_(r))of about 55,000 daltons (lane 3).

No reactivity was seen in extracts of E. coli lacking the plasmid (lane1). Furthermore, the expression of this fusion protein is inducible withisopropyl-beta-D-thiogalactopyranoside (IPTG) (compare lanes 2 and 3).Therefore, it is concluded that this long open reading frameencompassing 252-1871 encodes the M. tuberculosis 65KD residues 252-1871encodes the M. tuberculosis 65KD antigen. The phrases "540 amino acidresidue protein", "540 protein", "65KD protein" and "65KD proteinantigen" are used interchangeably herein for the 65KD protein of M.tuberculosis.

In addition, the purified recombinant 65KD protein was used in Westernblot analyses using serum from human patients known to be infected withM. tuberculosis . In preliminary studies, antisera from those patientsimmunoreacted with the purified recombinant protein.

Those studies illustrate the use of that natural or recombinant proteinas an antigen in a diagnostic assay method for the presence of naturallyoccurring antibodies to the 65KD protein in the infected patients, andthus for the detection of a Mycobacterium tuberculosis infection inthose patients. Similar results are obtained in a more usual solid phaseassay such as are carried out in a microtiter plate where therecombinant 65KD protein is affixed to a solid phase matrix to form asolid phase support and patient serum is the source of antibodies to beassayed.

Solid phase assays whether carried out in a microtiter plate, a dipstickor as a Western blot all require the similar steps and constitutevariants of each other. Each has a solid phase matrix (microtiter platewell, stick surface or nitrocellulose) to which the purified natural ora recombinant 540 amino acid protein coded for by the genome of M.tuberculosis as antigen is affixed, usually by adsorption, to form thesolid phase support. The assayed sample such as patient serum orcerebrospinal fluid (where evidence of tubucular meningitis is sought tobe assayed) in liquid form is admixed with the solid phase support toform a solid-liquid phase admixture. That admixture is maintained underusual biological assay conditions (e.g. zero degrees C to about 40degrees C) for a time period sufficient for any antibodies present inthe assayed sample to immunoreact with and bind to the antigen of thesolid phase support. The solid and liquid phases are separated as byrinsing. The presence of antibodies bound to the solid support isthereafter determined as with a labeled reagent that reacts with thebound human antibodies.

A labeled reagent that reacts with bound human antibodies present isadmixed with the solid phase to form a second solid-liquid phaseadmixture. That second solid-liquid phase admixture is maintained for atime period sufficient for the labeled reagent to react with the boundhuman antibodies. The second solid-liquid phase admixture is separatedas by rinsing, and the amount of label present is determined. An amountof label present above a background, control value indicates thepresence of anti-65KD protein antibodies and thus an infection by M.tuberculosis.

The labeled reagent that reacts with the bound human antibodies ispreferably a labeled preparation of xenogenic anti-human antibodies suchas alkaline phosphatase-conjugated goat anti-human Ig antibodies thatare available from Tago, Burlingame, CA. The presence of the boundalkaline phosphatase is typically determined spectrophotometrically bymeasurement of the enzymatic hydrolysis of a substrate molecule such asp-nitrophenyl phosphate to p-nitrophenol. Other enzymes such ashorseradish peroxidase and other label types such as radioactiveelements like iodine 125 are also useful. S. aureus protein A linked toa label such as ¹²⁵ I can also react with the bound human antibodies ofthe separated solid phases to detect their presence.

The above diagnostic assay method is typically carried out in a clinicalsetting using a kit. The kit comprises at least one package thatcontains a solid phase support having a purified 540 protein encoded bythe M. tuberculosis genome that is from the mycobacterium or is arecombinant protein as discussed herein affixed as an antigen to a solidmatrix such as a plastic microtiter plate or dipstick. One or moreadditional reagents such as the labeled reagent that reacts with solidphase-bound human antibodies, a substrate for the labeled reagent (whereneeded for the label), buffer salts in solution or dry form, and thelike can also be present in separate packages in the kit.

D. The 65KD Antigen Gene is Expressed in E. coli

Because previous studies had shown that most mycobacterial genes werenot expressed in E. coli using the mycobacterial transcription andtranslation signal sequences [Clark-Curtis et al., (1985), J.Bacteriol., 161:1093-1102; and Thole et al., (1985) Infect. Immun.,50:800-806] a protein expression library was used in the cloningstudies. In the λgt11 -M. tuberculosis library, the insertedmycobacterial coding sequences should be expressed as fusion proteinswith beta-galactosidase [Young et al., (1983) Proc. Natl. Acad. Sci.USA, 82 2583-2587]. It was somewhat surprising to find that the openreading frame encoding the 65KD antigen did not extend to the 5'-end ofthe mycobacterial DNA insert in λSK119. This suggested that the 65KDantigen was being expressed using the mycobacterial transcription andtranslation signal sequences.

With respect to the previously described E. coli consensus signalsequences, the mycobacterial sequences 180-230 base pairs upstream ofthe presumed initiator ATG codon do display reasonable matches with theconsensus sequences for the -35 (3/3 match with the highly conservedTTG) and -10 (4/6 match with TATAAT) regions of E. coli promoters[Rosenberg et al., (1979), Ann. Rev. Genet., 13:319-353]. There is alsoa 5/5 match with the Shine-Dalgarno sequence [Shine et al., (1974),Proc. Natl. Acad. Sci. USA, 71:1342-1346] for a prokaryotic ribosomebinding site (GGAGG) 13 base pairs upstream of the presumed initiatortriplet for the 65KD antigen open reading frame. Although the preciselocations of the mycobacterial regulatory sequences have not beendetermined experimentally, the results of the two studies describedbelow suggest that the mycobacterial sequences are indeed functional inE. coli.

The size of the anti-65KD reactive material produced by the recombinantswas determined in a Western blot assay. To do this, crude lysates ofcells expressing recombinant plasmids or phage that had been shown tocontain the entire 65KD antigen gene (λSK116, pTB12) as well as thosethat had been shown to contain a large portion of the 65KD antigen openreading frame fused to B-galactosidase (λRY3146; pTB22 that contains the540 protein DNA from position 438 through position 1948 of FIG. 2) wereprepared as described in the Materials and Methods section.

The lysates were electrophoresed on 10% Laemmli SDS-polyacrylamide gels,and the separated proteins were electrophoretically transferred tonitrocellulose. The SDS-denatured, immobilized proteins were thenreacted with monoclonal antibodies specific for the 65KD antigen.

The results using antibody IT-13 are shown in FIG. 4. In cellsexpressing recombinants carrying the fused open reading frame, themonoclonal antibodies detected a single strongly reactive speciesmigrating with an M_(r) of about 160,000 daltons as well as occasionallydetecting smaller species (FIG. 4, Panel B, lane 3). In another fusedopen reading frame recombinant, the monoclonal antibodies detected asingle reactive species migrating with an M_(r) of about 55,000 daltons(FIG. 4, Panel A, lane 3). In the extracts of the cells expressingrecombinants that contained the entire 65KD gene, the monoclonalantibodies detected a single strongly reactive species that migratedwith an M_(r) of about 64,000 daltons (FIG. 4, Panel B, lanes 1 and 21.

Smaller reacting species (about 40,000-55,000 daltons) were observedwhen large amounts of the extracts were loaded (lane 5) or when theprotease inhibitor was omitted from the lysis buffer. Occasionally, aminor reacting species was also observed migrating with an M_(r) ofabout 67,000 daltons.

Given the sizes of the anti-65KD-reactive materials, these data indicatethat the 65KD antigen can be expressed using the mycobacterialtranslation initiation signals present in the 65KD gene. Also, since thevector contribution to the recombinant plasmids does not contain anyknown sequences that are properly located and oriented to promote thetranscription of the inserted DNA, these data suggest that themycobacterial transcription initiation signals function in E. coli toallow the expression of the 65KD antigen.

In order to obtain an approximate measure of the efficiency ofutilization of the mycobacterial transcription and translationinitiation signals in E. coli , two plasmids were constructed thatplaced the expression of enzymatically active beta-galactosidase underthe control of either the mycobacterial signal sequences or the lac genesignal sequences present in the plasmid pUC19.

First, the 3000 bp BamHI fragment from pMC1871 that contains the codingsequences for amino acid residues 8-1021 of beta-galactosidase [Shapiraet al., (1983), Gene, 25:71-82] was inserted into the BamHI site ofpTB12 (residues 437-442 of the sequence presented in FIG. 2). Theresulting 8.1 kbp plasmid (pTB27) contains an open reading frame thatencodes a fusion protein with 63 amino acid residues derived from the65KD antigen gene followed by 1014 amino acids of beta-galactosidase,and whose expression is under the control of the transcription andtranslation signal sequences present in the mycobacterial DNA. Asexpected, this construct expresses a protein of about 120,000 daltonsthat reacted with anti-beta-galactosidase antibodies in a Western blotassay.

Second, the 3000 bp BamHI fragment from pMC1871 was inserted into theBamHI site in the polylinker of pTB9 that contains a 2.4 kbp fragment ofthe 65KD antigen gene inserted in the EcoRI site of pUC19. The resulting8.1 kbp plasmid (pTB28) contains an open reading frame that encodes afusion protein with 15 amino acid residues derived from the pUC19 lacZgene and polylinker sequences followed by the 1014 amino acid residuesof beta-galactosidase and whose expression is under the control of thelac gene signal sequences present in pUC19.

Crude extracts of cells containing these plasmids were assayed forbeta-galactosidase activity as previously described. In cells containingpTB27, beta-galactosidase activity [about 2800 units/microgram (ug)protein] was about one-fourth that (11,000 units/ug protein) found inIPTG-induced cells containing pTB28. Given the unknowns inherent in thisstudy (e.g., the specific activities and relative stabilities of the twofusion proteins), one cannot make a precise quantitative statement aboutthe relative strengths of the mycobacterial signal sequences and the E.coli lac gene signal sequences based on the relative enzymaticactivities found in the two cell extracts. However, the data do indicatethat these mycobacterial transcription and translation signal sequencesare efficiently recognized in E. coli.

E. The 65KD Antigen Sequence

Several interesting features of this long open reading frame have beenrevealed by a computer-aided analysis of the sequence. The overall basecomposition of this open reading frame is 65.5% G+C. However, the G+Ccontent varies considerably within the codons such that the G+C contentof the bases occupying the first two residues of the codons is 55% whileit is 87% for the bases found in the third position of the codons;thereby producing a bias towards using codons that have a G or C in thethird position.

For example, 50 of the 51 leucine codons (CTX) have a G or C in thethird position. Interestingly, the essentially random occurrence of anyof the four bases in the first two positions of a codon plus thepreference for G or C in the third position of a codon is one strategythat allows an organism to have a high G+C content without limitingaccess to the amino acids whose codons contain A or T residues in thefirst two positions.

Although the deduced amino acid residue sequence of the 65KD antigen isparticularly rich in alanine, glycine, leucine, and valine residues, theoverall amino acid residue composition contains 52% hydrophobic and 48%hydrophilic residues. Computer-aided analysis of the alpha helicalcontent Chou et al., (1978), Adv. Enzym., 47:45-148 and hydrophobicity[Hopp et al., (1981), Proc. Natl. Acad. Sci. USA, 78:3824-3828] of theamino acid residue sequence revealed numerous regions that couldparticipate in alpha helical structures and no extended regions of highhydrophobicity. These data suggest that the 65KD antigen is not anintegral membrane protein but rather its sequence resembles that of asoluble protein.

As discussed before, the 65KD antigen appears to be a major T cellimmunogen and antigen in man. It has been suggested that immunodominantT cell epitopes are short stretches of amino acids that can formamphiphilic helices where one side of the helix is hydrophobic and theother side hydrophilic, 1 Berzofsky, (1985), Science, 229:932-940. Basedon computer modeling, seven stretches of amino acids within the sequenceof the 65KD antigen have been identified that could form suchamphiphilic helices. A list of those peptides is shown in Table 2,below.

                  TABLE 2                                                         ______________________________________                                        Residue                                                                       Positions.sup.l                                                                          Sequence.sup.2                                                     ______________________________________                                         11-28  (58)                                                                             A R R G L E R G L N A L A D A V K V                                 66-79  (59)                                                                             E K I G A E L V K E V A K K                                        114-130 (60)                                                                             G L K R G I E K A V E K V T E T L                                  154-172 (61)                                                                             Q S I G D L I A E A M D K V G N E G V                              219-233 (23)                                                                             L L V S S K V S T V K D L L P                                      394-408 (62)                                                                             I E D A V R N A K A A V E E G                                      494-508 (63)                                                                             V K V T R S A L Q N A A S I A                                      ______________________________________                                         .sup.l Residue positions are denominated using the one letter amino           residue sequence of the 65KD protein shown in FIG. 2 that depicts the         methionine residue coded for by the triplet beginning at base pair            position 252 as the first residue of the protein. Parenthesized numbers       refer to peptide numbers that begin with petide number 1 shown in Table 4     .sup.2 These amino acid sequences are shown from left to right and in the     direction from aminoterminus to carboxyterminus, as is customary in the       art.                                                                     

F. DCH Assay With A Recombinant 65KD Protein

Exemplary delayed cutaneous hypersensitivity (DCH) assays were carriedout using illustrative recombinant proteins described herein as testantigens after immunization with M. tuberculosis, M. bovis or saline.These assays were carried out following the procedure described inMinden et al. (1986) Infec. Immun. 53:560-564.

Briefly, the mammalian hosts were immunized with a sufficient amount ofM. tuberculosis or M. bovis to induce an immunological response, or witha control (saline). After maintaining the animals for a time periodsufficient for the initial immunological response to the immunogen tosubside, the animals were challenged by intradermal injection withinocula containing the 65KD protein, a recombinant 65KD protein, or arecombinant fusion protein that contained the 65KD protein as the testantigen dissolved or dispersed in a physiologically tolerable diluent,or with a control. The test antigens were present in an amountsufficient to induce erythema and induration at the site ofadministration in a mammal previously immunized with M. tuberculosis orM. bovis.

The results of this study are shown in Table 3, below.

                  TABLE 3                                                         ______________________________________                                        DCH Assays With Recombinant Antigens                                                    No. Positive/No. Assayed                                                      Of Guinea Pigs Immunized With.sup.2 :                               Challenge   M.           M.                                                   Antigen.sup.l                                                                              tuberculosis                                                                              bovis  Saline                                        ______________________________________                                        Saline (0)  0/5          0/5    0/5                                           BNN97.sup.3 (10)                                                                          0/5          0/5    0/5                                           λ1089.sup.4 (10)                                                                   5/5          5/5    0/5                                           λ1089.sup.4 (1)                                                                    5/5          5/5    0/5                                           pTB22.sup.5 (10)                                                                          5/5          5/5    0/5                                           pTB22.sup.5 (1)                                                                           5/5          5/5    0/5                                           BCG-S.sup.6 (1)                                                                           5/5          5/5    0/5                                           PPd.sup.7 (5 T.U.)                                                                        5/5          5/5    0/5                                           ______________________________________                                         .sup.l Challenge antigen compositions were injected intradermally as          discussed in Materials and Methods using amounts of 1 or 10 ug/100 ul per     injection as indicated by the parenthesized numeral after each antigen,       except for purified protein derivative (PPd,) that was used in an amount      of 5 tuberculin units (T.U.).                                                 .sup.2 The number of guinea pigs exhibiting positive DCH responses is in      the numerator, whereas the number of guinea pigs assayed is in the            denominator. The immunization protocol is described in Materials and          Methods.                                                                      .sup.3 BNN97 was a crude lysate prepared from gtllinfected E. coli. The       crude lysate was partially purified by ammonium sulfate precipitation as      described in the Materials and Methods section.                               .sup.4 λ1089 was a crude lysate prepared from λSK119infecte     E. coli that expressed the 65KD antigen. The crude lysate was partially       purified by ammonium sulfate precipitation as described in the Materials      and Methods section.                                                          .sup.5 pTB22 was a crude lysate prepared from E. coli containing pTB22        that expressed the 65KD antigen as a fusion protein that contained a          portion of the betagalactosidase molecule and about the carboxyterminal 8     percent of the 65KD protein. The crude lysate was partially purified by       ammonium sulfate precipitation as described in the Materials and Methods      section.                                                                      .sup.6 6BCG-S was an extract of M. tuberculosis prepared as described in      the Materials and Methods section.                                            .sup.7 PPd was obtained from Connaught Laboratories, Ltd., Willowdale,        Ontario, Canada.                                                         

As can be seen from the above results, the 65KD protein coded for by theDNA sequence of FIG. 2 can be utilized in DCH as part of a method todetermine whether a mammalian host such as guinea pig had previousimmunological exposure to M. tuberculosis since the T leucocytes of thehost animals produced erythema and induration at the sites ofadministration in the animals previously immunized with M. tuberculosisand M. bovis, and produced no reactions in the saline-immunized animals.Those results also show that recombinant 65KD protein molecules aresimilarly useful. Recombinant fusion proteins that contain a portion ofthe beta-galactosidase molecule peptide-bonded to the amino-terminus ofthe 65KD protein are also useful, as are fusion proteins that contain aportion of the beta-galactosidase molecule and an immunologically activeportion, about the carboxy-terminal 85% of the 65KD protein, e.g., theprotein expressed by pTB22. Fusion proteins that contain one or morepeptide sequences as are described in Tables 2 and 4 hereinafter arealso useful. The phrase "previous immunological exposure" and itsgrammatical variants is used herein to mean that the mammalian host hadbeen immunized or infected by one of the mycobacteria and the hostmammal mounted an immune response (primary response) to the immunogensprovided by the mycobacteria, and that that immune response hadsubsided.

G The 517Amino Acid Protein

1. The Open Reading Frame

A second long open reading frame begins with an ATG codon at positions3948-3946 of FIG. 2 and extends to a TAA triplet at positions 2397-2395on the DNA strand complementary to the DNA strand encoding the 65KDantigen, thereby making those open reading frames adjacent in thegenome. This open reading frame can encode a protein that contains asequence of 517 amino acid residues, and that protein is referred toherein as the "517 amino acid protein" or the "517 protein". The 517protein coding region thus extends from position 3948 through position2398 of FIG. 2.

Given that the two long open reading frames are located adjacent anddownstream from each other on the complementary strands, one mightexpect that the transcription of one gene might interfere with thetranscription of the other unless there were transcription terminationsignals within the intergenic region. Indeed, there are several short

sequences (e.g., 2134-2160) within the 520 base pair intergenic regionthat have features reminiscient of the transcription termination signalsof gram-negative bacteria [Rosenberg et al., (1979), Ann. Rev. Genet.,13:319-353]. That is, regions containing short, G+C-rich, invertedrepeats capable of forming stem and loop structures followed by astretch of three or more T residues about 20 bases from the center ofdyad symmetry. Perhaps these inverted repeats might function astranscription termination signals to allow the independent expression ofeach of these mycobacterial genes.

To determine if the 517 amino acid open reading frame was expressed intoprotein in E. coli , extracts of cells containing a plasmid (PTB11)carrying the complete open reading frame were probed with a polyclonalrabbit antiserum elicited with a sonicated extract of M. tuberculosisbacteria in a Western blot assay. In these recombinants, the putativeprotein product of the 517 amino acid open reading frame would have tobe expressed using the mycobacterial regulatory sequences. Thepolyclonal antiserum detected more than 100 species in an extract of M.tuberculosis cells as well as the 65KD antigen in extracts of E. colicells carrying the appropriate plasmid (pTB12), but did not detect anynovel proteins in extracts of E. coli cells containing plasmids carryingthe 517 amino acid residue protein open reading frame. Hence, eitherthis open reading frame is not expressed in E. coli using themycobacterial regulatory sequences or the particular antiserum used inthe immunoblots did not contain antibodies directed against thisprotein.

It is not surprising that this open reading frame is not expressed in E.coli using the before-discussed recombinant since previous studiessuggest that most mycobacterial genes are not expressed in E. coli[Clark-Curtiss et al., (1985), J. Bacteriol., 161:1093-1102; and Tholeet al., (1985), Infect. Immun., 50-800-806]. Also, this open readingframe does not contain any impressive matches to the E. coli consensuspromoter sequences within the 400 bases upstream of the ATG tripletalthough it does contain a 3/5 match with the Shine-Dalgarno consensussequence for ribosome binding sites 12 oases upstream of the initiatorATG triplet. nonetheless, given the size of this open reading frame andits unique structural features (discussed below), it most likely isexpressed into protein in M. tuberculosis and can be expressed in E.coli using a recombinant vector designed for that expression, as isdiscussed hereinafter.

2. Structural Features of the 517 Protein

The second long open reading frame could encode 517 amino acids or aprotein of about 51,000 daltons (calculated M.W.=50,561). The deducedamino acid residue sequence is rich in alanine, asparagine, glycine, andserine and overall is composed of 54% hydrophobic residues and 46%hydrophilic residues. The amino acid sequence of this protein does notdisplay significant homologies with any of the protein sequences in theProtein Database.

The most striking features of this sequence occur between amino acidresidues 200 and 350, and more particularly at positions 217 through328. This region contains many repeats of short stretches of aminoacids.

For example, the five amino acid sequenceasparagine-asparagine-asparagine-isoleucine-glycine (N N N I G , usingone letter code) is repeated three times consecutively at positions 227through 241.

But perhaps the most interesting feature concerns a five amino residuesequence that displays at least partial matches with several sequencesin this region. These five residue sequence repeats begin at position217 and continue through position 328 of FIG. 2. The consensus sequenceof this repeat appears to be X-glycine-asparagine-Z-glycine, or XGNZG,using one letter code. For the fifteen sequences that match thisconsensus sequence, X is most often phenylalanine, serine or threonine(12/15), although X can also be isoleucine, leucine and aspartic acid. Zis most often isoleucine or threonine (10/15), but is also sometimesserine, leucine or valine. Additional sequences between positions 200and 350 display partial matches with the consensus sequence (i.e., match2 of the 3 core residues).

The above five residue sequences are arranged, from the amino-terminustoward the carboxy-terminus, with two abutting (contiguous) XGNZGsequences that are contiguous with the three NNNIG sequences that arethemselves contiguous to eight contiguous XGNZG sequences. A gap ofabout seventeen residues follows, that is itself followed by threecontiguous XGNZG consensus sequences. Another gap of five residuesensues that abuts another two contiguous five residue XGNZG consensussequences. Interestingly, both of those gaps contain sequences havingtwo of the three core residues of the consensus sequence, as well asproperly spaced X and Z residues.

It is further noted that this region contains a direct repeat of afourteen amino acid residue sequence with only one mismatch (residues295-308 and 315-328). Those sequences are shown below using one lettercode:

    ______________________________________                                        295-308   F N S G S G N I G F G N S G                                         315-328   F N S G S G N I G I G N S G.                                        ______________________________________                                    

As expected, since the amino acid residue repeats of the consensussequences are not exact, the nucleotide sequences in this region are notexact repeats. This observation suggests that recombinational processessuch as an unequal crossing over may not play a role in causing rapidevolutionary changes in this region as is often observed for highlyrepeated nucleotide sequences.

The remainder of this protein sequence does not display any otherparticularly striking features.

The highly repetitious nature of the 517 residue protein is reminiscentof the repeated structures found in the major coat proteins of thesporozoite stage of the malaria parasite [Nussenzweig et al., (1985),Cell, 42:401-403]. These circumsporozoite or CS proteins are 40-60 KDproteins located on the membrane of the infectious sporozoite andcontain a strongly immunodominant epitope that reacts with most of theanti-sporozoite antibodies found in polyclonal antisera as well as allof the monoclonal antibodies raised against the sporozoite stage. Thecentral region of these proteins contains 20-40 tandemly arrangedrepeats of a 11-12 amino acid sequence.

In Plasmodium falciparum, the immunodominant epitope is contained withinthree consecutive repeats of the sequenceasparagine-alanine-asparagine-proline (NANP; which is repeated 37 timesin one isolate) and antibodies directed against this 12-residue repeatcan provide immunologic protection against infection with the malariaparasite. The sequence of the repeat differs in the various species ofthis parasite and the number of repeats can vary within differentisolates of the same species. The similarity of the repeated nature ofthe CS protein and that of the 517 amino acid residue M. tuberculosisprotein raises the interesting possibility that the repeated sequencesin the 517 residue protein might play some role in the immune responseto mycobacteria

3. Expression of the 517 Protein

Although the 517 protein was not expressed using the before-describedrecombinant construct, that protein was expressed in E. coli using arecombinant expression vector designed specifically for its expression.That recombinant expression vector was constructed as follows, using thebase pair numbering of FIG. 2. It is to be understood that the DNAsequence of interest here is that shown in the lower of the two DNAsequences depicted, and that sequence, is read from right to left and inthe direction from 5'-end to 3'-end, although the sequence positionnumbers are read from left to right and in the direction from 5'-end to3'-end for the upper sequence.

The double stranded DNA sequence of FIG. 2 was cleaved with endonucleasePvuII to provide a fragment that extends from position 3511 to position4018 (509bp). That fragment was ligated into the SmaI site of the pUC19vector to form intermediate I. Two orientations were possible forligation of the PvuII fragment in the vector. Proper orientation wasdetermined by usual methods such as isolation of severalinsert-containing clones and preparation of restriction maps of the DNAfrom those clones. For example, a BglI fragment from a clone having thePvuII DNA fragment in the proper orientation contains about 1500 bp,whereas a BglI fragment from a clone having an improperly oriented PvuIIfragment contains only about 1300 bp. Intermediate I was introduced intoE. coli to propagate the vector DNA.

The propagated DNA of intermediate I was whereafter cleaved withendonucleases NotI (position 603) and SalI (in the pUC19 polylinkersite). The resulting NotI-SalI fragment was discarded, whereas theremainder of the DNA of Intermediate I was retained.

A further sample of the DNA sequence of FIG. 2 was cleaved withendonucleases NotI (position 3603) and SalI (position 2202) to provide aNotI-SalI fragment that was ligated into the appropriate sites of theretained Intermediate I DNA to form a second pUC19-derived vectordenominated intermediate II. That vector contained the complete 517protein DNA sequence, and was propagated further in E. coli.

The propagated DNA of Intermediate II was collected and cleaved withendonucleases EcoRI and HindIII at their respective sites in the 517protein gene and in the polylinker of pUC19. The resulting EcoRI-HindIII fragment that contained the 517 protein DNA was thereafter collectedand ligated into those respective sites in the polylinker of plasmidvector pKK223-3 to form Intermediate III that contained thecarboxy-terminal portion of the gene. Intermediate III was cloned in E.coli JM105. (pKK223-3 and JM105 are available from Pharmacia FineChemicals, Piscataway, N.J.)

A further sample of the DNA of Intermediate II was cleaved with EcoRIalone to excise a portion of that DNA from a position in the polylinkerto position 2969 in the 517 protein. The resulting EcoRI fragmentcontaining DNA that codes for the amino-terminal portion of the 517protein was collected, and was thereafter ligated into the single EcoRIsite of Intermediate III to form the expression vector that contains theentire 517 protein gene That vector was also cultured in E. coli JM105as a replication/expression medium.

It is noted that two orientations were possible for ligation of theEcoRI fragment in the expression vector. Proper orientation wasdetermined by usual methods such as isolation of severalinsert-containing clones and preparation of restriction maps of the DNAfrom those clones. For example, a KpnI-HindIII fragment from a clonehaving the EcoRI DNA fragment in the proper orientation contains about2000 bp, whereas a KpnI-HindIII fragment from a clone having animproperly oriented EcoRI fragment contains only about 800 bp.

Expression of a recombinant protein from vector pKK223-3 is induciblewith IPTG, and the induced recombinant protein is expressed as theprotein itself, and not as a fusion product. The resulting E coli cellswere thus grown and then induced with IPTG, as discussed elsewhereherein.

The expressed protein was produced in a relatively large amount andcould be readily identified in an SDS-PAGE gel from a lysate of the Ecoli cells. The 517 protein had an apparent M_(r) of about 55,000daltons in SDS-PAGE, as expected.

The expressed 517 protein can also be collected and purified, as with anaffinity column made from Sepharose 4B (Pharmacia) to which antibodiesraised to one or more of the 517 protein-related peptides are bound viathe cyanogen bromide activation technique, or by ammonium sulfateprecipitation, followed by DEAE-cellulose chromatography.

H. Recombinants and Vectors

The present invention thus contemplates the purified recombinant 540protein and 517 protein, as well as those recombinant fusion proteinsthat also include all or a portion of another molecule such asbeta-galactosidase fused to the amino-terminus of those proteins. Eachof those recombinant proteins is useful for inducing the production ofantibodies that immunoreact with those respective molecules as obtainedfrom M. tuberculosis itself or from cells infected with thatmycobacterium. Methods of preparing such antibodies are well known inthe art and are similar to the methods utilized for the peptides of thisinvention as described hereinafter.

The purified recombinant 540 amino acid residue protein or its fusionproteins when present in an effective amount in an inoculum are alsouseful in a DCH assay, as described before. Those proteins are alsouseful in diagnostic methods and kits useful for assaying for thepresence of infection by M. tuberculosis .

Nucleotide sequences are also contemplated, as are non-chromosomalplasmid vectors useful for propagating those DNA sequences andexpressing the protein products coded for by those sequences.

A nucleotide sequence of this invention consists essentially of one ofthe before-described sequences. Thus, a nucleotide sequence of theinvention excludes additional nucleotides that affect the basic andnovel characteristics of a nucleotide sequence that codes for the 540protein or the 517 protein.

A nucleotide sequence of the invention can include one or moretranscriptional promoter sequences operationally linked to the sequenceadjacent to the 5'-end thereof. Where translation of the DNA and proteinexpression are desired, the DNA also includes a translation initiatingcodon (ATG) and a translation terminating codon (TAA or TAG or TGA),each operationally linked adjacent to the 5'-end and 3'-end,respectively, of the sequence, with the translation initiating codonbeing located between the promoter sequence and the 5'-end.

A DNA sequence that codes for all or a portion of another molecule canalso be included in the DNA molecule so that the translated (expressed)proteinaceous molecule is a fusion protein that includes an amino acidresidue sequence of all or a portion of that other molecule fused(linked by a peptide bond) to the expressed 540 protein or 517 protein.An exemplary fusion polypeptide is the fusion protein molecule discussedherein that contains a portion of the beta-glactosidase molecule fusedto the amino-terminus of the 540 amino acid residue protein.

All of the nucleotide sequences shown in FIG. 2 can be present so longas an enumerated DNA molecule remains replicable, where only replicationis desired. Where replication and translation (proteinaceous moleculeexpression) are desired, those nucleotide sequences are present so longas the DNA molecule remains replicable and the proteinaceous moleculecontaining the amino acid residue sequence of 540 protein or 517 proteinexpressed exhibits immunological cross-reactivity with the antibodiesraised to an appropriate peptide described herein. In more preferredpractice, only those base pairs needed for expression of a desiredprotein are utilized.

A non-chromosomal, plasmid vector for propagation and expression of adesired DNA nucleotide sequence as defined herein in areplication/expression medium, e.g., a unicellular organism or the likesuch as E. coli, S. cerevisiae or mammalian cells such as COS cells, isalso contemplated. That vector comprises a replicon that is compatiblewith the relication/expression medium and contains therein the foreignDNA molecule (e.g., all or a portion of the sequence shown in FIG. 2}tobe replicated in a manner such that the vector can propagate the DNAmolecule.

In addition, the non-chromosomal plasmid vector also includes thosesequence components that are utilized for transcription and translation.To that end, a transcriptional promoter can be operationally linked tothe DNA molecule present adjacent to the 5'-end thereof, as alreadynoted. The transcriptional promoter can be endogenous to the vector orexogenous to the vector. A transcriptional promoter endogenous to thevector such as the lac Z promoter-operator utilized in the vectorsderived from pUC19 or the trp-lac (tac) promoter of pKK223-3 ispreferred. A translational terminator can also be operationally linkedadjacent to the 3'-end of the DNA molecule in some instances, althoughthe nucleotide sequence represented by the formula of FIG. 2 containssuch terminator sequences.

An initiation codon (ATG) adjacent to the 5'-end of the sequence thatbegins translation in a replication/expression medium is also requiredto be present in a vector used for expression. Such a codon can beprevent in a defined DNA molecule in frame, as is the case with thesequences shown in FIG. 2, or can be a portion of the precursor plasmidvector nucleotide sequence.

The before-discussed transcription promoter, translation initiating andtranslation terminating codons are frequently parts of thenon-chromosomal plasmid vector as compared to a DNA molecule of theinvention. For use in expression of the proteinaceous molecule, theprecursor plasmid frequently also includes a ribosome binding site(Shine-Delgardo sequence) adjacent to the 5'-end of the foreign DNAmolecule and located upstream from the initiation codon, as is wellknown. The vector's promoter such as the lacZ and tac promoters utilizedherein typically contain a ribosome binding site.

Thus, the nucleotide sequence of the plasmid vector used for expression,aside from those nucleotides needed for the replication and generalvector function include, in frame and from 5'-end to 3'-end, a ribosomebinding site operationally linked adjacent to the 5'-end of atranscription promoter; that promoter operationally linked to the 5'-endof the translation initiating codon; that codon operationally linked tothe 5'-end of: (a) a sequence of a portion of another molecule that isexpressed as a fusion protein with the desired protein, or (b) a foreignDNA molecule of this invention; where (b) is present, that sequence isoperationally linked to the 5-end of a DNA molecule of this invention.An expression vector containing the foreign DNA molecule of thisinvention, (however linked adjacent to its 5'-end) also contains atranslation terminating codon adjacent the 3'-end of the foreign DNA.

It is to be understood that all of the DNA sequences of the vector mustbe compatible with the replication/expression medium utilized forreplicating the DNA, and more preferably for expressing a product codedfor (encoded by) a DNA molecule of this invention.

It is also to be understood that the before-recited signal sequences ofthe useful vector can be supplied to that vector by the foreign DNA orby a precursor to the final vector. For example, the translationinitiation and termination codons in the expression vector for the 517protein are provided by the foreign DNA, whereas the promoter andribosomal binding site sequences are provided by the precursor plasmid.

A vector of the invention is at least capable of replicating(propagating) a DNA molecule of the invention. More preferably, thevector is capable of not only replicating a DNA molecule, but is alsocapable of expressing or translating the genomic information of that DNAinto a recombinant protein molecule that is immunologically similar tothe 540 protein or the 517 protein; i.e., will induce cross-reactiveantibodies.

A non-chromosomal plasmid vector of this invention need not be limitedto those vectors useful for replication and translation (expression) inE. coli as host replication/expression medium. Substantially any vectoruseful for replicating (propagating) and expressing a DNA sequence canbe utilized for replicating the DNA, e.g. in mammalian or eukaryoticcells.

A wide range of such vectors is commercially available as areappropriate host replication media. Exemplary vectors, both plasmids andbacteriophages and hosts are available from the American Type CultureCollection of Rockville, MD, and are listed in its CATALOGUE OFBACTERIA, PHAGES AND rDNA VECTORS, sixteenth ed., 1985. In addition,plasmids, cosmids and cloning vectors are listed as being available incatalogues from Boehringer Mannheim Biochemicals of Indianapolis, Ind.;Bethesda Research Laboratories, Inc. of Gaethersberg, Md., and NewEngland Biolabs, Inc. of Beverly, Mass.

Peptides

Another aspect of the present invention relates to a peptide thatconsists essentially of an amino acid residue sequence that correspondssubstantially to a portion of the 540 or the 517 protein sequence. Sucha peptide contains 5 to about 40 amino acid residues, and morepreferably about 10 to about 20 amino acid residues that correspondsubstantially in sequence to a protein of either the 540 amino acidresidue protein or the 517 amino acid residue protein that are coded forby the DNA sequence shown in FIG. 2.

A useful peptide most preferably contains only those amino acid residuesthat are identical or homologous to (conservative substitutions for)residues present in a sequence of either of the two above proteins.Additional residues of substantially any length can also be present ateither or both termini of the peptide. However, any additional residuesmust not interfere with the activity of the peptide, as discussedhereinafter, and therefore, a peptide of this invention is said to"consist essentially" of an enumerated sequence. For example, a peptideof the invention is free of immunosuppressing sequences. In addition, ifadditional residues are present, and together with an above peptidecorrespond substantially in sequence to further portions of the sameprotein to which the sequence of the peptide substantially corresponds,the resulting peptide is of a molecular weight less than that of thenaturally occurring 540 or 517 proteins, respectively.

A peptide of this invention is useful, inter alia, for inducing theproduction of antibodies in a laboratory mammal such as a mouse orrabbit. Those induced antibodies immunoreact with the inducing peptideas well as with the protein to which the peptide sequence substantiallycorresponds when that protein is in an SDS-denatured form as in aWestern blot analysis subsequent to SDS-PAGE analysis.

Thus, the anti-peptide antibodies can be used in solid phase assays forthe detection of the presence of an antigen that is the 540 protein orthe 517 protein of M. tuberculosis. In this instance, the assayed samplesuch as sputum provides the antigen that is affixed to the solid phasematrix to form the solid support. An aqueous composition containing theanti-peptide antibodies or their idiotypic portions (bindingsite-containing portions) is admixed, maintained and separated from thesolid phase as previously discussed for the presence of anti-65KDprotein antibodies. The presence of bound anti-peptide antibodies isthereafter assayed to determine the presence of the M. tuberculosisantigen in the sample, following the broad admixture, separation andanalysis steps previously described. Whole antibodies and theiridiotype-containing portions such as Fab and F(ab')₂ portions arecollectively referred to as paratoic molecules.

In exemplary studies, antibodies (paratopic molecules) were raised inNew Zealand white rabbits to both the amino-terminal andcarboxy-terminal polypeptide sequences (Peptides 1 and 54 of Table 4,hereinafter) of the 540 protein. Varying dilutions of pure M.tuberculosis cultures were bound to the walls of microtiter plates toform a solid support and one or the other of the two aqueousanti-peptide antibody preparations was admixed with the solid support toform a solid/liquid phase admixture. After maintaining the solid/liquidphase admixture for a time period sufficient for the anti-peptideantibodies to bind to the mycobacterial antigens present, the phaseswere separated. The solid phase was rinsed to assure removal of unboundanti-peptide antibodies. The presence of anti-peptide antibodies boundto the solid support was thereafter determined by standard methods.

As a result of those studies, it was determined that the presence of amycobacterial antigen could be detected at a concentration of about 10⁵organisms per well. Sputum samples from persons with active infectionsof M. tuberculosis typically contain about 10⁵⁻¹⁰ ⁶ organisms in thevolume of a sample utilized in the study. Thus, anti-peptide antibodiesraised to a peptide of this invention such as those raised to Peptides 1and 54 can be utilized to detect mycobacterial antigens present at alevel found in a clinical environment.

Antibodies were similarly raised to the immunologically activerecombinant, fusion, 540 protein produced by pTB22, and a similarantibody binding study was carried out. The results were generallysimilar to those discussed above, except that this assay was somewhatmore sensitive, presumably as a result of the polyclonal character ofthe induced antibodies.

In addition to the above assays for mycobacterial antigens, severaladditional immunoassays can be carried out using antibodies induced (a)by the previously-mentioned 540 protein, or more particularly (b) by animmunologically active portion thereof such as the fusion proteinproduced by pTB22, a fusion protein that contains a peptide sequence ofTables 2 or 4 fused to a portion or all of another molecule such asbeta-galactosidase, or by a peptide of Tables 2 and 4. Such additionalimmunoassays are well known in the art and include, for example, doubleantibody, "sandwich", assays, and competition assays as where a peptideor other antigen described herein competes for the antibodies with amycobacterial antigen in the assayed sample.

In each of the immunoassays, a sample to be assayed for the presence ofa mycobacterial 65KD cell wall protein-a antigen is admixed in anaqueous medium with paratopic molecules raised to the 540 protein, ormore particularly to an immunologically active portion thereof. Theresulting admixture is maintained for a time period sufficient for theparatopic molecules to immunoreact with mycobacterial antigens presentin the admixed sample to form an immunoreactant. The presence, andusually the amount, of immunoreactant formed is determined.

The anti-peptide paratopic molecules can themselves contain a label.Preferably, however, a second label-containing reagent is utilized thatreacts with the bound paratopic molecules such as whole anti-peptideantibodies. The peroxidase-conjugated goat-anti-mouse antibodiesutilized herein are exemplary of such reagents.

A solid phase assay kit that utilizes the anti-peptide antibodies orother paratopic molecules induced by an immunologically active portionof the 540 protein is also contemplated herein for clinical use of thebefore-described method. Here, the kit contains at least a solid phasematrix to which the assayed-for antigen of the sample or antibodies canbe affixed in one package and a preparation of anti-peptide oranti-immunologically active 540 protein portion paratopic molecules thatimmunoreact with the 540 (65KD) protein or the 517 protein in a secondpackage. Additional packages of reagents similar in type and function tothose previously mentioned can also be included.

For inducing paratopic molecules such as whole antibodies, a usefulpeptide is typically linked to an antigenic carrier molecule such askeyhole limpet hemocyanin (KLH) as a conjugate, the conjugate isthereafter dispersed in a physiologically tolerable diluent as aninoculum, and the inoculum is injected into the laboratory mammal usingwell known procedures. The inoculated animal is maintained and givenbooster injections as required, until a desired antibody titer to theinducing peptide is achieved The mammal's antibody-containing serum isthereafter obtained, purified as desired, and utilized in a diagnosticassay such as an SDS-PAGE/Western blot for the presence of asubstantially corresponding protein.

The word "inoculum" in its various grammatical forms is used herein todescribe a composition containing an amount of peptide conjugate,peptide polymer (as described hereinafter), 65KD protein or recombinantprotein sufficient for a described purpose that is dissolved ordispersed in an aqueous, physiologically tolerable diluent. Exemplarydiluents are well known and include water, physiological saline,phosphate-buffered saline, Ringer's solution, incomplete Freund'sadjuvant and the like.

Inocula can contain varying amounts of a preferred peptide or polymer,depending upon its use.

Where paratopic molecules are to be formed or an inoculum is otherwiseto be used as a vaccine, about 100-500 micrograms of peptide or peptidepolymer are used per injection into laboratory animals such as mice,rabbits or guinea pigs. Larger amounts are utilized for larger mammals,as is known. Similar amounts of peptide or polymer are utilized for invivo DCH assays.

Smaller amounts of antigenic peptide or antigenic peptide polymer areutilized for in vitro stimulation assays. Using 200 microliters (ul) oftotal volume and about 1-2×10⁴ PBMC and about 1×10⁵ antigen-presentingcells, concentrations of about 0.1 to about 50 micrograms of antigen permilliliter are useful.

Exemplary procedures for the chemical synthesis of a useful peptide aswell as preparation of a conjugate and use of the conjugate to raiseantibodies can be found in U.S. Pat. Nos. 4,636,463, 4,599,231,4,599,230, 4,545,931, 4,544,500, all of whose disclosures areincorporated herein by reference.

Another use for a preferred peptide of this invention is in an assay forthe presence of mycobacterially-exposed (or immune); i.e., previouslyimmunologically exposed, mononuclear cells such as T cells in a bodysample containing such cells. Mycobacterially-exposed (or immune)mononuclear cells are cells that themselves have been immunologicallyexposed to a mycobacterial immunogen or whose progenitor cells had beenso exposed to such an immunogen. Thus, a preferred peptide can be usedto determine whether a mammal has been immunized against a mycobacteriumor has or has had a mycobacterial infection.

In such an assay, peripheral blood mononuclear cells, and particularly Tcells, from the mammal are provided. Those cells are admixed andcontacted in an aqueous cell culture medium with a stimulating amount ofboth antigen presenting cells and a preferred peptide of the inventionto form a stimulatory cell culture. The stimulatory cell culture ismaintained for a period of time sufficient for immune mononuclear cellspresent to be stimulated and to evidence that stimulation, usually about18-96 hours and most usually 24-48 hours, under usual cell cultureconditions. The presence of mononuclear cell stimulation is thereafterdetermined.

Where mononuclear cell stimulation is found, it indicates the presencein the assayed mononuclear cell population of cells which themselveswere immunologically exposed to a mycobacterium or whose parental linewas immunologically exposed to a mycobacterium.

As is illustrated by the results shown in Table 3, hereinbefore, therecombinant 540 protein and recombinant fusion protein containing aportion of the beta-galactosidase molecule and an immunologically activeportion of the 540 protein were useful in stimulatingmycobacteriologicallyimmune mononuclear cells in vivo in a DCH assay.Such molecules can be utilized in the above-described assay, and in thestimulatory assays described hereinafter in the same manner as can thepeptides, and in the place of a peptide.

Mononuclear cell stimulation can be determined in a number of mannersthat are well known in the art, some of which are described specificallyhereinafter. The cells of the mononuclear cell population that mostusually are stimulated are T cells, and for that reason, the mononuclearcells will be usually referred to hereinafter as T cells. Moreparticularly, T cells that exhibit the CD4 or T4 (CD4⁺ or T4⁺ antigenand those that exhibit the CD8 or T8 (CD8⁺ or T8⁺) antigen are the cellsthat are typically stimulated. Those T cells are often more generallyreferred to as helper and killer or cytotoxic T cells, respectively.

Exemplary manners in which T cell stimulation can be determined include(a) proliferation as assayed by the uptake of a radiolabeled nucleosidesuch as [³ H]-deoxythymidine also referred to as [³ H]-Tdr, [³H]-thymidine ([³ H]-T), (b) secretion of interferon-gamma, (c) secretionof interleukin-2 (IL-2), (d) secretion of granulocyte macrophage-colonystimulating factor (GM-CSF), (e) cytotoxicity, a phenomenon that canoccur with T cells such as T4⁺ T cells as well as with T8⁺ cells, (f)the ability to provide an in vitro B cell helper function, (g) theability of immune T cell clones to provide a delayed cutaneoushypersensitivity (DCH) response in vivo as described herein and in U.S.Pat. No. 4,689,397 whose disclosures are incorporated by reference, and(h) the ability of immune T cell clones to provide protective immunityin vivo.

A kit is also contemplated for use with the immediately preceding assay.That kit can include a number of containers, at least one which containsa preferred peptide antigen of this invention or a polymer of such apeptide antigen whose repeating units are comprised of a"di-Cys-terminated" peptide as is described hereinafter. A mixture oftwo or more such preferred peptides or their polymers can also bepresent. A sufficient amount of a preferred peptide or peptide polymeris contained in the container to perform at least one assay using thatmethod.

The assay kit can further include a premeasured amount of buffer orother salt for the preparation of an inoculum of the peptide or polymerupon the addition of water or other suitable aqueous medium. Theinoculum can also be provided in premixed aqueous form either at theconcentration for use or as an aqueous concentrate to be diluted.

Of course, the particular constituents and concentrations of thoseconstituents can differ between in vitro and in vivo assays as well asbetween different mammals whose cells are to be assayed. Suchconstituents and concentrations can be readily determined by skilledworkers. It is to be further understood that a previously describedfusion protein that includes an immunologically active portion of amycobacterial 65KD cell wall protein-a antigen can also be used to theexclusion of a peptide or polymer thereof as the antigen. Thus, theantigen of the kit can more broadly be described as a mycobacterialantigen.

A useful peptide corresponds substantially in sequence to a sequence ofeither the 540 or the 517 proteins discussed previously. Substantialcorrespondence of peptide sequences can be determined in a number ofways.

Of course, two peptides having identical sequences correspondsubstantially, as do to peptides that share identical sequences but alsocontain one or more further sequences. Similarly, two sequences thatdiffer by conservative substitutions such as isoleucine for leucine orvaline, asparatic acid for glutamic acid, asparagine for glutamine,arginine for lysine, serine for threonine, phenylalanine for tryptophanand tyrosine for phenylalanine, also correspond substantially.

Two sequences can also correspond substantially when antibodies raisedto one immunoreact with another. For example, the particular peptidesdisclosed hereinafter can be used to raise antibodies that immunoreactwith the 65KD (540) protein, and consequently, those peptides correspondsubstantially in sequence to the sequence of the 65KD protein.

Biochemical evidence from immunoassay and from analogy with conservedprotein-protein interaction in solved X-ray crystallographic structureswith differing sequences such as in the dimer contacts of oligomericenzymes indicates that the conservation of protein-protein recognitiondoes not require a strict conservation of sequence, for relatedness.Whereas single amino acid residue changes may affect such recognition toa wide degree depending upon the nature of the substitution, in generalterms the relatedness and thus substantial correspondence of twodiffering amino acid sequences with respect to protein-protein (andantigenic and/or immunogenic) recognition can be expressed in terms ofseven basic amino acid residue parameters:

(1) hydrophobicity;

(2) evolutionary occurrence of changes in known sequences;

(3) size of side chain;

(4) charge and polarity;

(5) preference for turned secondary structure;

(6) preference for beta strand secondary structure; and

(7) preference for helical secondary structure.

To define the degree of sequence identity relevant to antigenic and/orimmunogenic recognition, and thus substantial correspondence of peptidevariants, a consensus matrix based upon the above seven criteria canalso be used to assign numerical values for each amino acid residue pairin the sequences being considered for substantial correspondence. Forthe purposes of the present invention, a consensus matrix developed byDr. Elizabeth Getzoff and Dr. John Tainer of the Scripps Clinic andResearch Foundation of La Jolla, Calif. can be used. That consensusmatrix is as follows, wherein the individual amino acid residues aredesignated by a one-letter code in the interests of conciseness:

    __________________________________________________________________________    A    R  N  D  C  Q  E  G  H  I  L  K  M  F  P  S  T  W  Y  V                  __________________________________________________________________________    A 7  -5 -1 -2 0  0  -1 2  -1 0  1  -2 2  -1 0  0  0  -3 -3 1                  R -5 10 0  -1 -3 2  -1 -5 5  -4 -4 5  -3 -2 -3 0  0  -1        -1                                                                  -4                       N -1 0  6  3  1  3  0  1  3  -2 -2 2  -1 -3 1  4  2  -3        0                                                                   -2                       D -2 -1 3  7  -2 1  4  0  0  -3 -3 0  -2 -4 0  1  0  -5        -2                                                                  -3                       C 0  -3 1  -2 7  1  -2 1  0  0  0  -2 0  0  0  3  4  -2        2                                                                   0                        Q 0  2  3  1  1  6  2  -1 4  0  0  2  0  0  0  1  3  -1        0                                                                   0                        E -1 -1 0  4  -2 2  7  -3 1  -3 -2 0  -1 -3 -2 0  0  -5        -3                                                                  -3                       G 2  -5 1  0  1  -1 -3 8  -2 -3 -3 -2 -2 -5 2  3  1  -6        -2                                                                  -2                       H -1 5  3  0  0  4  1  -2 8  -1 0  4  0  0  0  1  2  0         2                                                                   -1                       I 0  -4 -2 -3 0  0  -3 -3 -1 5  4  -3 2  2  -2 -2 0  0         0                                                                   4                        L 1  -4 -2 -3 0  0  -2 -3 0  4  6  -2 4  3  -1 -2 0  1         0                                                                   3                        K -2 5  2  0  -2 2  0  -2 4  -3 - 2                                                                              8  -1 -3 -1 0  0  -4        -2                                                                  -3                       M 2  -3 -1 -2 0  0  -1 -2 0  2  4  -1 6  2  0  -1 0  0         -1                                                                  2                        F -1 -2 -3 -4 0  0  -3 -5 0  2  3  -3 2  7  -2 -3 0  4         3                                                                   2                        P 0  -3 1  0  0  0  -2 2  0  -2 -1 -1 0  -2 7  2  1  -4        -1                                                                  -1                       S 0  0  4  1  3  1  0  3  1  -2 -2 0  -1 -3 2  5  3  -3        0                                                                   -1                       T 0  0  2  0  4  3  0  1  2  0  0  0  0  0  1  3  6  -2        1                                                                   0                        W -3 -1 -3 -5 -2 -1 -5 -6 0  0  1  -4 0  4  -4 -3 -2 9         2                                                                   0                        Y -3 -1 0  -2 2  0  -3 - 2                                                                              2  0  0  -2 -1 3  -1 0  1  2         8                                                                   0                        V 1  -4 -2 -3 0  0  -3 -2 -1 4  3  -3 2  2  -1 -1 0  0         0                                                                   5                        __________________________________________________________________________

Sequence comparison using the foregoing consensus matrix involves thedetermination of all possible alignments and the subsequent scoring ofthese alignments by the matrix. Two sequences are then aligned bycomputing the maximum match score from the consensus matrix. Analignment score in standard deviation units can be determined by takingthe difference between the maximum matched score and the average maximummatched score for random permutation of the two sequences, and thendividing by the standard deviation of the random score.

For the present purposes, a consensus matrix score greater than threestandard deviations (approximately an average value of about 3 perresidue) shows significant relatedness at a confidence level of morethan 99.7%. This is a restrictive criterion since it gives a frequencyof 0.005 for all 5-residue peptides and 0.0014 for all 13-residuepeptides occurring in 2222 known protein sequences. Similarly, aconsensus matrix score greater than two standard deviations(approximately an average value of about 2 per residue) showssubstantial correspondence to be significant at a confidence level ofmore than 95.4%.

To determine substantial correspondence for the purposes of the presentinvention, the consensus matrix score is calculated by ascertaining thematrix value for each aligned amino acid residue pair underconsideration, and then summing the individual values for each suchpair. The obtained sum is then compared against the number of standarddeviations signifying the desired confidence level. If the obtained sumis greater than the product of the selected number of standarddeviations times the number of amino acid residue pairs underconsideration, then the amino acid residue sequences being comparedcorrespond substantially to the indicated confidence level.

For example, to ascertain the substantial correspondence of the aminoacid residue sequences

    --Lys--Trp--Phe--Cys--Gly--and

    --Arg --Ile--Phe --Cys--Gly--the consensus matrix yields the following values

    ______________________________________                                                         Value                                                        ______________________________________                                        Lys- & -Arg- or K & R                                                                            5                                                          Trp- & -Ile- or W & I                                                                            0                                                          Phe- & -Phe- or F & F                                                                            7                                                          Cys- & -Cys- or C & C                                                                            7                                                          Gly- & -Gly- or G & G                                                                            8                                                          Total              27                                                         ______________________________________                                    

For substantial correspondence at the 99.7% confidence level, theconsensus matrix score must exceed the number of amino acid residuepairs under consideration times 3; i.e., 5×3 or 15. Inasmuch as 27 isgreater than 15, substantial correspondence is indeed present for theabove two peptide sequences.

For the purposes of the present invention, substantial correspondenceamong peptides within the scope of the invention preferably is presentat least to about 95% confidence level, and more preferably to at leastabout 99% confidence level.

A DNA sequence can correspond substantially to another DNA sequence ifboth sequences contain sequences of fifteen bases that are in phase andidentical, or bases that are not identical but code for an identicalsequence of amino acid residues, or code for amino acid residuesequences that correspond substantially. Thus, amino acid residuesequences that correspond substantially are encoded by DNA sequencesthat correspond substantially.

It is to be noted that two or more peptide sequence can substantiallycorrespond as determined by one or both of the latter two definitionsand still exhibit different immunoreactivities with antibodies raised tothe intact protein as is found in nature or with T cells stimulated bysuch natural proteins. An example of this phenomenon is discussedhereinafter.

In addition to the specific peptides disclosed in Table 2, hereinbefore,further peptides that correspond in sequence to a portion of the 540protein sequence are also useful herein. A list of those peptides isprovided in Table 4, below.

                  TABLE 4                                                         ______________________________________                                        Peptides                                                                      Peptide                                                                       Number Residues.sup.1                                                                         Sequence.sup.2                                                ______________________________________                                         1      1-15    M A K T I A Y D E E A R R G L                                  2     11-25    A R R G L E R G L N A L A D A                                  3     21-35    A L A D A V K V T L G P K G R                                  4     31-45    G P K G R N V V L E K K W G A                                  5     41-55    K K W G A P T I T N D G V S I                                  6     51-65    D G V S I A K E I E L E D P Y                                  7     61-75    L E D P Y E K I G A E L V K E                                  8     71-85    E L V K E V A K K T D D V A G                                  9     81-95    D D V A G D G T T T A T V L A                                 10      91-105  A T V L A Q A L V R E G L R N                                 11     101-115  E G L R N V A A G A N P L G L                                 12     111-125  N P L G L K R G I E K A V E K                                 13     121-135  K A V E K V T E T L L K G A K                                 14     131-145  L K G A K E V E T K E Q I A A                                 15     141-155  E Q I A A T A A I S A G D Q S                                 16     151-165  A G D Q S I G D L I A E A M D                                 17     161-175  A E A M D K V G N E G V I T V                                 18     171-185  G V I T V E E S N T F G L Q L                                 19     181-195  F G L Q L E L T E G M R F D K                                 20     191-205  M R F D K G Y I S G Y F V T D                                 21     201-215  Y F V T D P E R Q E A V L E D                                 22     211-225  A V L E D P Y I L L V S S K V                                 23     219-233  L L V S S K V S T V K D L L P                                 24     231-245  L L P L L E K V I G A G K P L                                 25     241-255  A G K P L L I I A E D V E G E                                 26     251-265  D V E G E A L S T L V V N K I                                 27     261-275  V V N K I R G T F K S V A V K                                 28     271-285  S V A V K A P G F G D R R K A                                 29     281-295  D R R K A M L Q D M A I L T G                                 30     291-305  A I L T G G Q V I S E E V G L                                 31     301-315  E E V G L T L E N A D L S L L                                 32     311-325  D L S L L G K A R K V V V T K                                 33     321-335  V V V T K D E T T I V E G A G                                 34     331-345  V E G A G D T D A I A G R V A                                 35     341-355  A G R V A Q I R Q E I E N S D                                 36     351-365  I E N S D S D Y D R E K L Q E                                 37     361-375  E K L Q E R L A K L A G G V A                                 38     371-385  A G G V A V I K A G A A T E V                                 39     381-395  A A T E V E L K E R K H R I E                                 40     391-405  K H R I E D A V R N A K A A V                                 41     401-415  A K A A V E E G I V A G G G V                                 42     411-425  A G G G V T L L Q A A P T L D                                 43     421-435  A P T L D E L K L E G D E A T                                 44     431-445  G D E A T G A N I V K V A L E                                 45     441-455  K V A L E A P L K Q I A F N S                                 46     451-465  I A F N S G L E P G V V A E K                                 47     461-475  V V A E K V R N L P A G H G L                                 48     471-485  A G H G L N A Q T G V Y E D L                                 49     481-495  V Y E D L L A A G V A D P V K                                 50     491-505  A D P V K V T R S A L Q N A A                                 51     501-515  L Q N A A S I A G L F L T T E                                 52     511-525  F L T T E A V V A D K P E K E                                 53     521-535  K P E K E K A S V P G G G D M                                 54     526-540  K A S V P G G G D M G G M D F                                 ______________________________________                                         .sup.1,2 See Notes 1 and 2 of Table 2.                                   

Preferred peptides for use in the previously described assay for thepresence of mycobacteriallyimmune mononuclear cells are those that arenumbered as follows, wherein the numbers are those shown in one or bothof Tables 2 and 4, are those shown in positions in the 540 protein aregiven in parentheses: Peptide 22 (211-255); Peptide 23 (219-233);Peptide 24 (231-245); Peptide 30 (291-305); Peptide 46 (451-465);Peptide 58 (11-28); Peptide 59 (66-79); Peptide 60 (114-130 ); andPeptide 62 (394-408).

Several proliferative assays were conducted using the peptides of theinvention. Results of those studied are shown and discussed below.

One study was carried out using pooled peripheral blood mononuclearcells (PBMC) from M. bovis BCG-vaccinated humans. The details of thisstudy are described in the Materials and Methods Section. Briefly, PBMCwere isolated and seeded into culture plate wells. Such PBMC populationscontain their own endogenous antigen-presenting or feeder cells. Apeptide of the invention was added as antigen at either 0.1 microgramper milliliter (ug/ml), 1 ug/ml or 10 ug/ml of culture. The antigen/cellculture mixture was maintained for a time period of six days, at whichtime, radiolabeled thymidine was admixed. The cultures were harvestedeighteen hours later and the thymidine incorporation was measured in aliquid scintillation counter. The results of this study are shown inTable 5, below.

                  TABLE 5                                                         ______________________________________                                        Protein 540                                                                   Peptide-Induced PBMC Proliferation.sup.l                                      Peptide       Residue  Proliferation                                          Number.sup.2  Positions.sup.3                                                                        Response.sup.4                                         ______________________________________                                        10             91-105  -                                                      21            201-215  -                                                      22            211-225  ++                                                     24            231-245  ++                                                     25            241-255  -                                                      30            291-305  ++                                                     35            341-355  -                                                      43            421-435  -                                                      46            451-465  +++                                                    47            461-475  -                                                      48            471-485  -                                                      49            481-495  -                                                      53            521-535  -                                                      54            526-540  ++                                                      58*          11-28    ++                                                      59*          66-79    ++                                                      60*          114-130  +++                                                     61*          154-172  -                                                       23*          219-233  +++                                                     62*          394-408  +++                                                     63*          494-508  -                                                      ______________________________________                                         .sup.1 Proliferation as measured by incorporation of [H]-thymidine in         counts per minute (cpm).                                                      .sup.2 Peptide number as shown in Tables 2 and 4.                             .sup.3 Peptide residue sequence positions as shown in Tables 2 and 4 and      in FIG. 2.                                                                    .sup.4 Proliferative response reported at the optimal peptide                 concentration is represented as follows: "+++" = 10,000-40,000 cpm; "++"      2000-10,000 cpm; or "-" =300-700 cpm. Proliferation in the absence of         peptide antigen was 421 ± 37 cpm, and was 82,857 ± 2,913 cpm in the     presence of an extract of M. tuberculosis. Standard deviations did not        exceed 15 percent in any of the triplicate measurements.                      *Peptides predicted to form amphiphilic helices.                         

The above results indicate that nine of the twenty petides assayedelicited a strong proliferative response. Thus, nine regions of the 540protein were identified as human T cell antigens.

Seven regions of the 540 protein were predicted by computer-assistedanalysis to form amphiphilic helices. Regions that can form amphiphilichelices appear to have a higher probability of being recognized by Tcells. Berzofsky, (1985) Science, 229:932-940. However, only five ofthose seven peptides provided a proliferative response. This indicatesthat amphiphilicity is neither sufficient nor necessary for a peptide tointeract with T cells.

In further studies with PBMC from individual BCG-immunized humans, aninfluence of HLA type was noted on reactivity. Thus, lymphocytes fromtwo persons with the HLA-DR4 allele reacted with Peptide 62 (positions364-408) but not with Peptide 30 (positions 291-305), whereas cells fromthree persons with the HLA-DR1 allele reacted with Peptide 30 but notwith Peptide 62.

The above results indicate a genetic, HLA restriction on theproliferation response. Thus, a mixture of preferred peptides or theirpolymers is preferred when assaying an out bred population such ashumans so that false negative responses can be minimized.

Another proliferation study was carried out with sixteen of the abovepeptides. The proliferating cells here were either one of two T cellclones or a polyclonal T cell line. One T cell clone came from atuberculosis patient (AH) and is designated K8AH. The second T cellclone was obtained from a person (JM) vaccinated with heat-killed M.leprae and is referred to as A7JM. The polyclonal T cell line (JM) wasalso obtained from the cells of JM that were initially stimulated withM. bovis BCG, stored frozen and thereafter stimulated with a recombinant65KD protein from M. tuberculosis (Oftung et al., (1987) J. Immunol.,138:927-931].

Proliferation was again assayed by the [³ H]-thymidine incorporationmethod. Here, autologous PBMC irradiated to inhibit proliferation butsufficiently viable to act as antigen-presenting cells were added to thecultures of both isolated T cell clones and the cell line along with amycobacterial antigen. After three days of maintenance, the cultureswere pulsed with the radiolabel for four hours, harvested and thencounted.

The details of this study are provided in the Materials and MethodsSection. The results are shown in Table 6, below.

                  TABLE 6                                                         ______________________________________                                        Protein 540                                                                   Peptide-Induced Proliferation.sup.1                                           Peptide.sup.2                                                                            Proliferative Response.sup.3                                       Number     K8AH       A7JM      JM                                            ______________________________________                                        59*        0.3  ±0.1                                                                             0.2 ± 0.1                                                                            0.3 ± 0.2                                  10         0.4 ± 0.2                                                                             0.1 ± 0                                                                              0.2 ± 0                                    21         0.3 ± 0 0.1 ± 0                                                                              0.2 ± 1                                    22         0.5 ± 0 14.0 ± 2.0                                                                           88.2 ± 15.8                                23*        0.2 ± 0.1                                                                             0.1 ± 0.1                                                                            9.0 ± 2.8                                  24         7.4 ± 0.3                                                                             0.1 ± 0                                                                              4.1 ± 0.1                                  25         0.9 ± 0.2                                                                             0.1 ± 0                                                                              0.3 ± 0.2                                  30         0.4 ± 0.1                                                                             0.1 ± 0                                                                              0.7 ± 0.2                                  35         0.4 ± 0.1                                                                             0.4 ± 0                                                                              0.3 ± 0.1                                  62*        0.4 ± 0.3                                                                             0.2 ± 0                                                                              0.3 ± 0.1                                  43         0.3 ± 0.1                                                                             0.1 ± 0.1                                                                            0.2 ± 0.1                                  46         0.5 ± 0.1                                                                             0.5 ± 0                                                                              11.4 ± 0.2                                 49         0.5 ± 0.1                                                                             0 ± 0  0.4 ± 0.3                                  53         0.1 ± 0 0.2 ± 0.1                                                                            0.2 ± 0.1                                  54         0.4 ± 0.2                                                                             0.2 ± 0.1                                                                            0.9 ± 0.1                                  63         0.4 ± 0 0.2 - 0.1 0.4 ± 0.1                                  --Ag.sup.4 0.5 ± 0.2                                                                             0.3 ± 0.2                                                                            0.4 ± 0.2                                  M. tuberculosis                                                                          4.3 ± 0.5                                                                             12.3 ± 1.9                                                                           30.1 ± 2.6                                 rec. 65KD.sup.5                                                               M. tuberculosis.sup.5                                                                    8.5 ± 1.6                                                                             11.5 ± 1.5                                                                           118.7 ± 5.4                                M. bovis BCG.sup.5                                                                       9.4 ± 0.7                                                                             21.6 ± 2.3                                                                           183.4 ± 19.0                               M. leprae.sup.5                                                                          0.5 ± 0.1                                                                             24.2 ± 2.0                                                                           119.7 ± 15.5                               ______________________________________                                         .sup.1,2 See notes of Table 5.                                                .sup.3 Proliferative responses, in cpm × 10.sup.- 3 ± standard       derviation for two or three replicate studies using 10 ug/ml of each          peptide. Positive values are underlined.                                      .sup.4 Response in the absence of antigen.                                    .sup.5 Affinity-purified recombinant M. tuberculosis 65 KD protein at 50      ug/ml and whole mycobacteria at 20 ug/ml.                                

The results shown in Table 6 illustrate the clonal specificity forantigens of the screened peptides. Thus, T cell clone K8AH, specific tothe M. tuberculosis complex [Oftung et al., (1987) J. immunol.,138:927-931] exhibited a proliferative response upon stimulation with aninoculum containing Peptide 24 (231-245). The T cell clone A7JM, whichshows cross-reactivity to M. tuberculosis and M. leprae, responded tostimulation by admixture and contact with an inoculum containing adifferent segment of the 65KD (540 protein) represented by Peptide 22(211-225), but not to inocula containing the flanking and overlappingPeptides 21 (201-215) and 23 (219-233).

The JM polyclonal T cell line also proliferated in response tocontacting with inocula containing Peptides 24 and 22. That cell linealso showed a significant proliferation in response to admixture andcontact with an inoculum containing Peptide 23, whose sequence overlapsboth of those sequences of Peptides 24 and 22, and that was predicted toform an amphiphilic helix. Contacting the polyclonal T cell line with aninoculum containing Peptide 46 (positions 451-465) also providedsignificant stimulation.

The genomic sequence of the 65KD protein of M. leprae and the putativetranslation product of that gene have been published. [Mehra et al.,(1986) Proc. Natl. Acad. Sci. USA, 83:7013-7017.] A comparison of the65KD protein amino acid residue the two sequences to be very similar,with only a relatively few different residues between them.

T cell clone A7JM had previously been shown to proliferate in responseto stimulation by both whole M. leprae and whole M. tuberculosis.[Mustafa et al., (1986) Lepr. Rev. Suppl., 2:123-130.] Consistent withthose findings, clone A7JM also proliferated when admixed and contactedwith an inoculum containing Peptide 64, below, whose sequence differedfrom that of Peptide 22 by the conservative change of the residue atposition 215 from glutamic acid of Peptide 22 to aspartic acid.

(22) A V L E D P Y I L L V S S K V

(64) A V L E E P Y I L L V S S K V.

T cell clone K8AH is able to discriminate between M. tuberculosis and M.leprae presented for stimulation as whole bacilli, and was similarlyable to exhibit the same discrimination at the peptide level. Thus, theM. tuberculosis-related Peptide 24 (231-245) could be used to stimulateclone K8AH, whereas contact with an inoculum containing Peptide 65,below, having the analogous M. leprae sequence did not stimulate thatclone to proliferate. Inocula containing Peptide 65 also did notstimulate clone A7JM or polyclonal cell line JM.

(24) L L P L L E K V I G A G K P L

(65) L L P L L E K V I Q A G K S L.

As can be seen from a comparison of the above sequences of Peptides 24and 65, those peptides differ by the substitution of two residues neartheir carboxy-termini. The glycine (G) at position 240 of Peptide 24 issubstituted as a glutamine (Q) in Peptide 65, and the proline (P) atposition 244 of Peptide 24 is substituted as a serine (S) in Peptide 65.

Thus, recognition of Peptide 24 by clone K8AJ must be influenced by oneor both of glycine-240 and proline-244. Interestingly, an inoculumcontaining Peptide 25 (241-255), which contains proline-244, did notcause stimulation of clone K8AH cells when admixed and contacted withthose cells.

A blocking study was carried out to determine whether an inoculumcontaining Peptide 65 could inhibit the stimulatory response caused byPeptide 24 on cells of T cell clone K8AH. Those studies showed that theM. leprae-related Peptide 65 could not block the response induced by theM. tuberculosis-related Peptide 24. This finding again implies thecriticality of one or both of the residues at positions 240 and 244 ofPeptide 24.

Further stimulation studies were carried out using M. leprae-related andM. tuberculosis pepides and the before-mentioned T cell clones and cellline. An inoculum containing Peptide 23 caused stimulation of thepolyclonal cell line. The sequence of that peptide is identical in bothM. leprae and M. tuberculosis. (See also Table 6.)

In addition, two M. leprae-related Peptides, 64 and 66, that eachcontain a single amino acid residue substitution as compared to theiranalogous M. tuberculosis-related Peptides, 22 and 46, respectively,also were capable of eliciting stimulation of M. leprae-immune cell lineJM when inocula containing one or the other were admixed and contactedwith those cells. Neither M. leprae-related Peptide 64 nor Peptide 66stimulated cells at T cell clone K8AH. The sequences of Peptide 66 andof its analogous Peptide 46 are shown below.

(46) I A F N S G L E P G V V A E K

(66) I A F N S G M E P G V V A E K.

Each of Peptides 64, 65 and 66 corresponds substantially to Peptide 22,24 and 46, respectively. That substantial correspondencenotwithstanding, the results above illustrate that there can bedifferences in reactivities of such peptides at the T cell level.

That different reactivities in T cell stimulation were found forsubstantially corresponding peptides that differed in sequence is notparticularly surprising in view of the type of interaction thought to beinvolved in T cell stimulation by an antigen as compared to anantigen-antibody interaction.

Thus, an antigen-antibody interaction is usually considered to be arelatively simple ligand-receptor interaction in which substitutions ofpolar for polar (e.g., glutamic for aspartic in Peptides 22 and 66) orapolar for apolar of about the same size (e.g., leucine for methioninein Peptides 46 and 67) typically are not of great consequence. Indeed,it has been shown that for some influenza-related 13-residue peptides,drastic substitutions can occur with little differences being noted inbinding by a monoclonal antibody raised to the parent peptide. See, forexample U.S Pat. No. 4,631,211.

T cell stimulation, on the otherhand, is thought to resemble a sandwichin which the T cell and the antigen-presenting or feeder cell are thebread and the antigen is the filling. Thus, a peptide antigen must bindto two receptors, one on the T cell, and the other, believed to be oneor more proteins of the major histocompatibility complex (MCH), on thefeeder cell. In addition, the binding between antigen and each of the Tcell and feeder cell receptors is thought to be weaker than is the usualantigen-antibody binding. It was not therefore surprising that theglycine to glutamine and proline to serine substitutions betweenPeptides 24 and 65 resulted in differences in T cell stimulation.

As noted previously, T cell stimulation can be manifest in a number ofmanners. The previous discussion has centered primarily on in vitro andin vivo proliferation assays. The results discussed below using T cellclones K8AH and A7JM illustrate further manifestations of T cellstimulation, and manners in which such stimulation can be determined.

Standard assays for secretion of IL-2, granulocyte macrophage-colonystimulating factor (GM-CSF) and interferon-gamma secretion into thesupernatants of aqueous stimulatory T cell cultures were conducted usingthe above cloned T cells to illustrate stimulation. Cytotoxicity againstmacrophages pulsed with the same stimulatory peptide or wholemycobacteria was also assayed. Details of these studies are provided inthe Materials and Methods Section. The results are shown in Table 7,below.

                                      TABLE 7                                     __________________________________________________________________________    Protein 540 Peptide-Induced                                                   Stimulatory Responses In T Cell Clones.sup.l                                                            IFN-  Cytotoxi-                                     T cell clones.sup.2                                                                     IL-2  GM-CSF.sup.4                                                                            Gamma.sup.5                                                                         city.sup.6                                    __________________________________________________________________________    K8AH + APC                                                                              <0.2  11 + 12 (9%)                                                                            10    ND.sup.7                                      K8AH + APC +                                                                            9.4 ± 0.6                                                                        153 ± 30 (>100%)                                                                     56    86.5 ±  0.6                                Peptide 24                                                                    K8AH + APC +                                                                            7.6 ± 0.2                                                                        87 ± 35 (71%)                                                                        44    85.8 ±  2.9                                M. tuberculosis                                                               A7JM + APC                                                                              0.2   5 ± 8 (4%)                                                                           5 ± 1                                                                            ND                                            A7JM APC +                                                                              6.8 ± 0.6                                                                        135 ± 4 (>100%)                                                                      63 ± 7                                                                           82.7 ± 10                                  Peptide 22                                                                    A7JM + APC +                                                                            6.8 ± 0.1                                                                        160 ± 14 (>100%)                                                                     40    84.7 ± 3                                   M. tuberculosis                                                               __________________________________________________________________________     .sup.1 T cell clones were stimulated by the peptide antigen shown             hereinbefore to specifically activate each clone. Stimulation was assayed     by four methods. T cells and antigenpresenting cells (APC) without antige     were used as negative controls in all assays. Results are expressed as th     mean ± standard deviation (where calculated) of duplicate of triplicat     cultures.                                                                     .sup.2 Culture contents in addition to the medium are shown in each entry     with plus signs (+) indicating the presence of mixed cellular components      and antigen (peptide or M. tuberculosis), where present.                      .sup.3 Results expressed in units per ml.                                     .sup.4 Assay based on three independent studies usinf three different bon     marrow donors. results are expressed in colonyforming units of GM per 2       × 10.sup.5 cells. Parenthesized percentages relate to the number of     colonies induced by a GMCSF positive control supernatant.                     .sup.5 Results expressed international units per ml.                          .sup.6 Results expressed as percentages as discussed in the Materials and     Methods Section. APC + antigen was used as a negative control.                .sup.7 ND = not done.                                                    

The results of Table 7 illustrate further standard techniques that areuseful in determining the presence of stimulated T cells in addition tothe proliferation assays discussed before.

Assays of T cell clones K8AH and A7JM indicated that both showed thehelper/inducer (T4³⁰ ,T8⁻) phenotype. Cells of the T4⁺ phenotype areprimarily helper cells that recognize antigen plus class II HLAproteins. Such cells are also known to exhibit cytotoxicity as is shownin Table 7.

Tuberculosis is a disease in which the cellular portion of the immuneresponse is involved to the substantial exclusion of the humoral(antibody) portion of the immune response. Thus, the ability of thepreferred peptide antigens to stimulate the T cell clones to not onlyproliferate but to also secrete IL-2, GM-CSF, and interferon gamma, eachof which constitutes a portion of the cellular immunity response,indicates that those peptides, their polymers, and mixtures thereof, aswell as the 540 protein (65 KD protein) can play an important role inprotective immunity.

That role in cellular immunity is underscored by the macrophagecytotoxicity exhibited by the clones stimulated by the peptides or thewhole mycobacteria. Similar cytotoxicity for other mycobacteria-reactiveT cell clones has been reported. [Mustafa et al., (1987), Clin. Exp.Immunol., 69:255-262; Kaufman et al. (1986), Lep. Rev. 57, Suppl.2:101-111.] However, it is believed that the above results are the firstdemonstration that the same sequence of one protein antigen are involvedin both T cell help and cytotoxicity. the in vivo role of such T4⁺cytotoxic T cells is believed to destroy those macrophages that havebecome incompetent to kill their intracellularly-growing mycobacteria.

A preferred peptide was previously described herein as being capable ofstimulating mycobacterially-immune mononuclear cells, and such a peptidewas said to be useful in assaying for present or prior immunologicalexposure of such cells to mycobacteria. A particularly preferred peptideor its polymer is also capable of immunizing an animal for protectionagainst mycobacterial infection such as M. tuberculosis.

Thus, the present invention also contemplates a vaccine againstmycobacteria such as M. tuberculosis that comprises a physiologicallytolerable diluent containing as immunogen an immunizing effective amountof (i) a peptide whose amino acid residue sequence correspondssubstantially to a particularly preferred T cell-stimulating peptidedescribed herein or (ii) a polymer of such a particularly preferred Tcell stimulating peptide as described herein.

Exemplary particularly preferred peptides include Peptides 22 and 24.Further particularly preferred peptides are those whose sequencescorrespond substantially to a sequence of the M. tuberculosis 540protein or another mycobacterial 65 KD protein and contain 5 to about40, more preferably about 10 to about 20 residues, and still further arecapable of stimulating proliferation of mycobacterially-immune, and fora tuberculosis vaccine, M. tuberculosis-immune, T cells that exhibit theT4⁺ and/or T8⁺ phenotype.

Further particularly preferred peptides can be obtained by following aprocedure similar to that discussed previously. Polyclonal T cells fromone or more individuals are contacted with an inoculum containing apeptide such as one of those of Tables 2 and 4, and more particularlypeptides such as those of Tables 5 and 6 that have already been showncapable of stimulating proliferation of mycobacterially-immune T cells.The peptides inducing proliferation are noted and the proliferating Tcells are cloned by the limiting dilution technique as described byOftung et al.,. (1987) J. Immunol., 138:927-931. The phenotypes of theproliferated T cells are determined as with the OKT series of monoclonalantibodies available from Ortho Diagnostic Systems, Inc. of Raritan,N.J. One or more of the peptides capable of causing proliferation of Tcells having the T4⁺ and/or

T8⁺ phenotype is utilized in the vaccine.

More preferably, a mixture of peptides, polymers having such peptides asrepeating units, or a polymer whose repeating units are a mixture ofsuch peptides that cause proliferation of T4⁺ and/or T8⁺ T cells isused. The reason of this preference stems from the already noted MHCrestriction. In addition, there is usually found an MHC restrictionbetween T4⁺ and T8⁺ T cells, the former recognizing antigen plus classII MHC protein and the latter recognizing antigen plus class I MHCprotein.

Peptides that correspond substantially to portions of the 517 proteinare also useful herein, and are defined as to substantial correspondencesimilarly to those peptides discussed before. The peptides substantiallycorresponding to a sequence of the 517 protein can contain as few asfive residues and are therefore somewhat shorter than are the shortestof the peptides discussed before.

Three peptides (denominated 55, 56 and 57) and their variantssubstantially correspond to sequences, written from left to right in thedirection from amino-terminus to carboxy-terminus and using one lettercode, having the formulas

(55) N N N I G,

(56) X G N Z G, and

(57) F N S G S G N I G F(I) G N S G

wherein X is an amino acid residue selected from the group consisting ofF, S, T, L, D and T; Z is an amino acid residue selected from the groupconsisting of T, I, L, S and V; and the parenthesized residue canreplace the residue shown to its left in the sequence. Thus, in peptide57, F and I are alternative residues. More preferably, X is selectedfrom the group consisting of F, S and T; and Z is selected from thegroup consisting of T and I.

Using the before-described consensus matrix to calculate whether thevariant pentapeptides defined hereinbefore by the consensus sequenceXGNZG correspond substantially, one finds that all of those variantscorrespond substantially at least at 99% confidence level. This can bereadily seen by determining the greatest differences caused bysubstitutions, then calculating the resultant consensus matrix score,and comparing that value to 3 times the number of residues compared, 5,(3×15=15).

Thus, for the X residue, substituting an Ile (I) for an Asp (D) residue,or a Ser (S) for a Phe (F) provides a value of -3 from the matrix.Similarly for Z, substitution of Ile (I) for Ser (S) or Ser (S) for Val(V) provides a value of -2 from the matrix. Since two Gly (G) residuesand the Asn (N) residues are present in any of the before comparedconsensus pentapeptide sequences, the presence of those residuesprovides a score of 22 (8+6+8=22). Subtraction of five [(-3)+(-2)] forthe above substitutions from 22 provides a total score for the comparedpentapeptides of 17.

Since 17 is greater than 15, any of the above substitutions to theconsensus sequence provides pentapeptides that correspond substantiallyat least at the 99% confidence level. Furthermore, since the abovesubstitutions caused the greatest numerical difference in the totalscore, any other of the before-discussed substitutions for both X and Zin the consensus sequence produces a total score; i.e., where X is Thror Leu and Z is Thr or Leu, in the consensus sequence produces a totalscore that is larger than 17, and consequently, all of thosepentapeptides also correspond substantially to each other at least atthe 99% confidence level.

Peptides 55 and 56 are typically utilized as one of a plurality ofrepeating units of a polymer having a relatively low molecular weight;i.e., less than about 10,000 daltons in weight. The smallest suchpolymer, or oligomer, contains two of the five residue peptides(pentapeptides) bonded together through a peptide bond formed betweenthe carboxy-terminal residue of a first pentapeptide repeating unit andthe amino-terminal residue of a second pentapeptide repeating unit.

For example, Peptide 57, above, can be viewed as a polymer or oligomerhaving two such pentapeptide repeating units bonded together by apeptide bond, and also containing an additional four residues at theamino-terminus of the oligomer.

Similar calculations can also be carried out for variants of the otherpeptides disclosed herein as one means of determining whether a peptidewith a different sequence from one of those specifically enumeratedcorresponds substantially to a specifically enumerated peptide, or to aportion thereof. For the purposes of epitope-paratope interactions,sequences containing at least five residues are the shortest sequencesthat should be compared since at least five or six residues appear to berequired for epitope-paratope interaction. See for example, Elder et al.(1987) J. Virol. 61:8-15; Atassi (1975) Immunochemistry 12:423-438; andBenjamini et al. (1969) Biochemistry 8:2242-2246.

Similarly, the sequence in isolated form

N N N I G N N N I G N N N I G that is also present at nucelotidepositions 3270

through 3226 of FIG. 2 can be considered a polymeric or oligomerictrimer of the sequence of peptide 55. Likewise, an isolated form of thesequence from nucleotide position 3210 through position 3107 can beviewed a polymer or oligomer that contains eight XGNZG pentapeptidesrepeated. Each of above polymers or oligomers contains a plurality ofthe pentapeptide repeating units bonded together by peptide bonds.

Solid phase peptide synthesis techniques, as are described in thebefore-discussed U.S. Patents whose disclosures are incorporated hereinby reference, are typically the most useful means of preparation foroligomers and polymers containing up to a total of about forty totalresidues (eight repeating pentapeptide units).

Genetic engineering techniques as are described herein are particularlyuseful for preparing larger polymers that contain more than about eightpentapeptide repeating units. For example, a double stranded DNAmolecule having the sequence shown in FIG. 2 from nucleotide position2959 through nucleotide position 3303, and in phase with the illustratedamino acid residue sequence of protein 517 can be excised from thelarger molecule shown in FIG. 2 or synthesized from appropriatedeoxyribonucleic acid derivatives using known techniques, and thereafterligated into an appropriate plasmid vector for expressing a peptidepolymer that corresponds substantially in sequence to the polymercontaining the pentapeptide repeating units shown beneath the sequenceat those positions in FIG. 2.

Higher molecular weight polymers; i.e., with average molecular weightsof about 10,000 to 1,000,000, or more, containing one or more of theabove 540 protein or 517 protein pentapeptide repeating units can alsobe prepared by oxidatively polymerizing a peptide that is terminatedwith cysteine (Cys; C) residues, or a "diCys-terminated" peptide. Theresulting polymer thereby contains its repeating units bonded togetherby oxidized cysteine (cystine) disulfide bonds.

For example, each of the before-discussed 540 protein peptides or 517protein pentapeptides can be synthesized to contain an additional Cysresidue at each of the amino- and carboxy-termini to providediCys-terminated peptides in their reduced forms. After synthesis, in atypical laboratory preparation, 10 milligrams of the diCys peptide(containing cysteine residues in un-oxidized form) are dissolved in 250milliliters (ml) of 0.1 molar (M) ammonium bicarbonate buffer. Thedissolved diCys-terminated peptide is then air oxidized by stirring theresulting solution gently for period of about 18 hours in the air, oruntil there is no detectable free mercaptan by the Ellman test. [SeeEllman, Arch. Biochem. Biophys., 82:70-77 (1959).]

The polymer so prepared contains a plurality of the synthetic, peptiderepeating units that are bonded together by oxidized cysteine (cystine)residues. Such polymers typically contain their peptide repeating unitsbonded together in a head-to-tail manner as well as in head-to-head andtail-to-tail manners; i.e., the amino-termini of two peptide repeatingunits can be bonded together through a single cystine residue as can twocarboxyl-termini since the linking groups at both peptide termini areidentical.

A 517 protein pentapeptide repeating unit can itself be contained in theform of an oligomer containing up to about eight pentapeptide repeatingunits, or in a shorter peptide such as the 14-residue Peptide 57. Stillfurther, a genetically engineered polypeptide such as that prepared fromthe DNA sequence of nucleotides at positions 2959 through 3303 that hasbeen further engineered to include condons for Cys (TGT or TGC) at the5'-and 3'-ends can also be polymerized.

The molecular weight of such a polymer can be controlled through theaddition of chain-terminating reagents. Exemplary chain terminatingreagents are cysteine itself and a peptide such as a before-describedpentapeptide that further includes a single Cys residue, preferably at aterminus.

The full names for individual amino acid residues are sometimes usedherein as are the well-known three letter abbreviations. One letterabbreviations (code) is also utilized. The Table of Correspondence,below, provides the full name as well as the three letter and one letterabbreviations for each amino acid residue named herein (See, forexample, L. Stryer, Biochemistry, 2nd ed., W. H. Freeman and Company,San Francisco, (1981), page 16). The amino acid residues utilized hereinare in the natural, L, form unless otherwise stated.

    ______________________________________                                        Table of Correspondence                                                                         Three letter One letter                                     Amino acid        abbreviation symbol                                         ______________________________________                                        Alanine           Ala          A                                              Arginine          Arg          R                                              Asparagine        Asn          N                                              Aspartic acid     Asp          D                                              Asparagine or aspartic acid                                                                     Asx          B                                              Cysteine          Cys          C                                              Glutamine         Gln          Q                                              Glutamic acid     Glu          E                                              Glycine           Gly          G                                              Histidine         His          H                                              Isoleucine        Ile          I                                              Leucine           Leu          L                                              Lysine            Lys          K                                              Methionine        Met          M                                              Phenylalanine     Phe          F                                              Proline           Pro          P                                              Serine            Ser          S                                              Threonine         Thr          T                                              Tryptophan        Trp          W                                              Tyrosine          Tyr          Y                                              Valine            Val          V                                              ______________________________________                                    

III. MATERIALS AND METHODS A. Recombinant studies 1. Bacteria, Phage andPlasmids

The E. coli strains used in this work were BNN97 [Young et al., (1983)Science, 222:778-782; ATCC 37194]; JM83 [Yanisch-Perron et al., (1985),Gene, 33:103-119; also ATCC 35607]; JM101 [Yanisch-Perron et al.,(1985), Gene, 33:103-119; also ATCC 33876]; Y1089 [Young et al., (1983),Science, 222:778-782; also ATCC 37196]; and Y1090 [Young et al., (1983),Science, 222:778-782; also ATCC 37197]. Plasmids pUC19 [Yanisch-Perronet al., (1985), Gene, 33:103-119] and pMC1971 [Shapira et al., (1983),Gene, 25:71-82] were purchased from Pharmacia Fine Chemicals,Piscataway, N.J. The recombinant DNA library of M. tuberculosis genomicDNA fragments in the λgt11 vector was constructed by R. Young et al.(1985), Proc. Natl. Acad. Sci. USA, 82:2583-2587, and made availablethrough the World Health Organization's Program for Research in theImmunology of Tuberculosis. Recombinanat phage λRY3143 and λRY3146 weregenerously provided by R. A. Young [Whitehead Institute, M.I.T.; Younget al., (1985), Proc. Natl. Acad. Sci. USA, 2 2583-2587]. Subclones ofthe mycobacterial DNA inserts in these recombinant phage wereconstructed in pUC19 or M13mp9 [Messing et al., (1982), Gene, 9:269-276;M13mp9 is listed for sale in the August, 1983 catalog of BethesdaResearch Laboratories, Inc.] vectors using standard recombinant DNAtechniques [Maniatis et al., (1982), Molecular Cloning-a laboratorymanual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.].

2. Antisera

Monoclonal antibodies specific for the 65KD antigen were obtained fromthe Immunology of Tuberculosis Scientific Working Group under a grantfrom the WHO/World Bank/UNDP Special Program for Vaccine Development.These antibodies included IT-13 [WTB-78; Coates et al., (1981), Lancet,2:167-169]; IT-31 [SA2D5H4; T. Buchanon, unpublished] and IT-33 [MLIIH9;Gillis et al., (1982), Infect. Immun., 37:172-178].Anti-beta-galactosidase antibodies were purchased from Cooperbiomedical,Malvern, Pa. Polyclonal rabbit antisera directed against a sonicate ofM. tuberculosis strain H37Rv were elicited as previously described[Minden et al., (1984), Infect. Immun., 46:519-525].

3 Immunoscreening of λgt11-M. tuberculosis Library

Clones reactive with the monoclonal antibodies specific for the 65KDantigen were isolated essentially as described by Young et al. [Young etal., Proc. Natl. Acad. Sci. USA, 82:2583-2587]. Briefly, for each 150 mmLB plate, 0.6 ml of a fresh overnight culture of Y1090 cells wereinfected with 1-2×105⁵ plaque-forming units (pfu) of the library. After3.5-4 hours of growth at 42° C., the plaques were overlaid with a drynitrocellulose filter that had been saturated with 10 millimolar (mM)isopropyl-beta-D-thiogalactopyranoside (IPTG; available from SigmaChemical Co.). The plates were incubated an additional 3.5-4 hours at37° C and then removed to room temperature and the position of thefilters marked.

The filters were washed briefly in TBST [50 mM Tris-HCl, pH 8, 150 mMNaCl, 0.05% Tween 20 [polyoxyethylene (20) sorbitan monolaurate]] andthen incubated in TBST plus 20% fetal calf serum. After 30 minutes atroom temperature, the filters were transferred to TBST plus antibody.

For the initial screen, the antibody mix contained a 1:1000 dilution ofadmixed IT-13, IT-31, and IT-33. The filters were incubated with theantibody solution overnight at 4° C. with gentle agitation, washed inTBST and reacted with biotinylated goat anti-mouse immunoglobulin, theVectastain ABC reagent, and developer as described by the manufacturer(Vector Laboratories, Burlingame, Calif.). After the color haddeveloped, the filters were washed with several changes of water and airdried.

Phage corresponding to positive signals were twice plaque-purified. Todetermine which monoclonal antibodies reacted with which of therecombinant phage, about 100 pfu of a purified phage stock wereinoculated in a small spot on a lawn of Y1090 E. coli

on an LB (Luria-Bertani broth) plate. The phage were allowed to grow andinduced to synthesize the foreign proteins as described above. Thefilters were then reacted with a 1:1000 dilution of one of themonoclonal antibodies. The subsequent steps were the same as for theinitial screen.

4. Western Blot Assays

Cells containing phage or plasmids in which the expression of theforeign sequences was under the control of the E. coli lac generegulatory sequences were induced to synthesize the foreign proteins byincubating the cells in the presence of 2.5 mM IPTG for 2 hours. Crudelysates of cells expressing λgt11 recombinants were made as described inHuynh et al; (1985), DNA Cloning Techniques: A Practical; Gover, ed ,IRL Press, Oxford, Vol. I, pp. 49-78. Briefly, those lysates were madeby harvesting cells from overnight cultures and resuspending the cellsin 10 mM Tris pH 7.5, 10 mM EDTA containing 100 ug lysozyme/ml. After 10minutes at room temperature, sodium dodecyl sulfate (SDS) was added to afinal concentration of 0.5%. A protease inhibitor (Trasylol, BoehringerMannheim, Indianapolis, Ind.) was added to all crude lysates at a finalconcentration of 15 0.03%-0.3%.

The crude protein preparations were electrophoresed on 10%polyacrylamide-SDS Laemmli gels [Laemmli, (1970) Nature, 227:680-685],and the separated proteins electrophoretically transferred tonitrocellulose [Towbin et al., (1979), Proc. Natl. Acad. Sci. USA,76:4350-4354]. The immobilized proteins were reacted with a 1:1000dilution of monoclonal antibody IT-13 in TBST overnight at 4° C. Thenitrocellulose filters were then washed, reacted withperoxidase-conjugated goat anti-mouse immunoglobulin, and developed aspreviously described [Niman et al., (1983), Proc. Natl. Acad. Sci. USA,80:4949-4953].

5. Nucleic Acid Sequencing

The sequences of 5'-end-labeled restriction fragments of themycobacterial DNA were determined by a modification of the partialchemical degradation technique of Maxam and Gilbert [Brow et al.,(1985), Mol. Biol. Evol., 2:1-12; and Maxam et al., (1976), Proc. Natl.Acad. Sci. USA, 74:560-564]. For the M13/dideoxy sequencing studies,Sau3AI fragments from the mycobacterial DNA inserts were subcloned intothe BamHI site of M13mp9. Phage DNA was isolated from the M13recombinants and subjected to the dideoxy chain termination sequencingreactions [Biggin et al., (1983), Proc. Natl. Acad. Sci. USA,80:3963-3965; and Sanger et al., (1980), J. Mol. Biol., 143:161-178].The products of the sequencing reactions were electrophoresed on 6%acrylamide/7M urea/0.5-2.5×TBE gradient sequencing gels, [Biggin,(1983), Proc. Natl. Acad. Sci. USA, 80:3963-3965]. The gels were driedunder vacuum and exposed to Kodak XRP-1 film. The nucleotide sequenceswere determined independently for both strands of the mycobacterial DNA.

Computer-aided analyses of the nucleic acid sequences and deducedprotein sequences were performed using the databases and programsprovided by the Nucleic Acid and Protein Identification Resources of theNational Institutes of Health as well as the programs of Chow et al.,(1978) Adv. Enzym., 47:45-148 and Hopp and Woods [Hopp et al., (1981),Proc. Natl. Acad. Sci. USA, 78:3824-3828].

6 Beta-galactosidase assays

Cells were grown in B broth or B broth plus 2.5 mM IPTG to an opticaldensity at 600 nanometers (OD₆₀₀) of about 0.3. Crude lysates were made,and beta-galactosidase was activity assayed as described by Miller(1972), Experiments in Molecular Genetics, Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y.

7. Capacity of Recombinants to Elicit DCH a. DCH Assays

Studies were carried out to determine whether the recombinant proteinsor purified protein derivative (PPd) (Connaught Laboratories, Ltd.,Willowdale, Canada) would elicit DCH reactions in Hartley guinea pigsthat had been immunized with sonicates of M. tuberculosis, M. bovis orsaline. Groups of guinea pigs were given three weekly intramuscular(i.m.) injections of sonicates suspended in incomplete Freund's adjuvant(IFA) as the physiologically tolerable diluent. Each injection contained1.0 milligram (mg) of protein. Some animals received a fourth injectionso that one week after the final injection, all animals were testedintradermally (i.d). Test antigens included the crude and partiallypurified recombinant extracts as well as saline and PPd as controls.Test antigens were used at 1-10 ug diluted in 100 ul ofphosphate-buffered saline at a pH value of pH 7.0 (PBS), containing0.0005% Tween 20 as the physiologically tolerable diluent. Groups ofunimmunized guinea pigs were similarly tested. All i.d. injections wereadministered into shaved areas on guinea pig flanks. Reactions were readat 24, 48 and 72 hours, and were considered positive when the diametersof erythema and indurated areas exceeded 10 mm.

b. Preparation of Crude Lysates

E. coli containing a plasmid or lambda phage of interest were grown byincubation at 37 degrees C. with aeration in B-broth to late phase inwhich absorbance at 600 nanometers (A₆₀₀) was between approximately 0.4and 0.6. IPTG was then added to a final concentration of 10 mM and thebacteria were further incubated for two hours.

The bacterial culture was then chilled on ice for 10 minutes and thecells were harvested by centrifugation at 6000 rpm for 10 minutes. Theresulting cell pellet was washed once in TBS (50 mM Tris, pH 8, 150 mMNaCl) by resuspension and recentrifiguation, and was thereafterresuspended (Sigma Chemical Co., St. Louis, Mo.) in a volume of TBS with0.5 molar sucrose equivalent to 1/10 the original culture volume.Lysozyme was added to the resulting resuspended solution to a finalconcentration of 50 ug/ml, and that admixture was incubated for 10minutes at 37 degrees C. Cells were harvested by centrifugation and wereresuspended in an equal volume of TBS. Thereafter, DNAse, Trasylol andSDS (Sigma) were added to the resulting admixture such that the finalconcentrations were 1 ug/ml, 0.1% and 1%, respectively. That admixturewas further incubated at room temperature for a time period of 10minutes with periodic mixing to effect completion of cell lysis. Theresulting crude lysate was stored at -20 degrees C. until use.

c. Partial Purification of Expressed 65KD Protein

Proteins containing the 65KD antigens were partially purified from crudelysates of E. coli expressing that protein by differential ammoniumsulfate precipitation. To that end, a crude lysate was first combinedwith a solution of saturated ammonium sulfate (SAS) to give a finalconcentration of 30% of the original lysate concentration. Precipitationwas effected as is well known in the art, and the resulting supernatewas retained The supernate was then combined with SAS to give aconcentration of 50% of that of the original lysate, and precipitationeffected again. The resulting pellet was retained, resuspended in PBSand dialysed against PBS. This resulting dialysed material is referredto as partially purified.

d. Preparation of Extracts of M. tuberculosis

M. tuberculosis strain H37Rv and M. bovis strain BCG were obtained fromthe culture collection of the National Jewish Hospital and ResearchCenter, Denver, Colo., and grown as previously described [Minden et al.,(1972) Science, 176:57-58 and Minden et al., (1972) Infect. Immun.,6:574-582].

Bacteria were then heat-killed and broken by sonication with ultrasonictreatment until, by microscopic examination, greater than 95% of thecells were disrupted. These disrupted bacteria were then subjected toultracentrifugation at 200,000×g for a time period of 2 hours, and thesupernate was retained. The supernates so obtained are referred to asH37Rv-S and BCG-S, respectively, and their antigenic and biologicalcharacteristics have been described.

B. Peptide Studies 1. Mycobacterial antigens

Armadillo derived killed M. leprae was supplied by Dr. R. J. W. Rees,Mill Hill in London from the IMMLEP (WHO) bank. M. tuberculosis and M.bovis BCG were kindly donated by Dr. Eng, National Institute of PublicHealth, Oslo, Norway. Bacilli were killed by irradiation (2.5 m rad).Recombinant M. tuberculosis 65 KD antigen, expressed from λgt11 as abeta-galactosidase fusion protein, were purified from E. coli lysatesprepared as described in Oftung et al., (1987) J. Immunol., 138:927-931on a high affinity anti-beta-galactosidase column (Promega Biotech,Madison, U.S.A.).

2. Synthetic peptides

The protected peptide resins were prepared by usual Merrifield solidphase techniques in groups of 100 by the method of Simultaneous MultiplePeptide Synthesis [Houghten, (1985) Proc. Natl. Acad. Sci. USA,82:5131-5135; and Houghten et al , (1985) Inter. J. Pept. Prot. Res.,27:673-678], and were cleaved twenty-four at a time in a newmulti-vessel apparatus [Houghten et al., (1986) Biotechniques,4:522-529]. Each synthesis resulted in the generation of 50-75 mg ofpeptide. Typical purities of the crude peptides ranged from 65-95%.

3. T-cell clones and lines

The T-cell clone K8AH from a tuberculosis patient (AH) and the T-cellclone A7JM from a killed M. leprae-vaccinated person (JM) wereestablished by the limiting dilution technique as described [Oftung etal., (1987) J. Immunol., 138:927-931]. The T-cell line was raised fromperipheral blood mononuclear cells (PBMC) of the donor JM by stimulationof 2×10⁶ PBMC/ml in complete medium (RPMI 1640+15% AB serum+1%penicillin and streptomycin) with M. bovis BCG (20 ug/ml) in 24-wellCostar plates. After 6 days of incubation at 37° C. in an atmosphere of5% CO₂ and 95% air, antigen-reactive cells were expanded by adding 100U/ml recombinant IL-2 two times per week. After long term storage inliquid nitrogen, T cells were propagated in vitro by stimulation of2×10⁵ cells/ml in 24 well Costar plates with whole bacilli as antigen(20 ug/ml) in the presence of 10⁶ irradiated autologous PMBC as feeder(antigen-presenting) cells and recombinant IL-2 (100 U/ml). Efficientexpansion of clones and lines was achieved by stimulation with antigenand feeder cells once and IL-2 twice per week. Determination of T cellsubsets was performed as previously described [Oftung et al., (1987) J.Immunol., 138:927-931].

4. Peptide-Induced T Cell Clone Stimulation Assays

The following assays were carried out for the inventors by Dr. FrederikOftung, at the Laboratory for Immunology, Norwegian Radium Hospital,Oslo, Norway. Initial assays for T cell stimulation were carried outusing coded samples.

a. Antigen-Induced Proliferation of T-cell Clones and Lines

Clonal (1×10⁴) or polyclonal (2×104) T cells and irradiated autologousPBMC (1×10⁵) were distributed to each well of 96-well flat bottom Costarplates Mycobacterial antigens as whole bacilli, recombinant antigens asaffinity-purified material or synthetic peptides were then added intriplicates or duplicates. The total culture volume was kept at 200microliters (ul).

After 72 hours of incubation, the cultures were given a 4 hour pulse of0.045 mBq [³ H]-thymidine (specific activity=185×10³ mBq/mM). Cells werethen harvested and radioactivity incorporated was determined by liquidscintillation counting [Mustafa et al., (1983) Clin. Exp. Immunol.,52:29-371.

The results are expressed as mean (triplicates or duplicates) values ofcounts per minute (cpm). Cells were considered to be proliferating inresponse to a given antigen where cpm in cultures with antigen minus cpmin cultures without antigen was more than 1000 and cpm in cultures withantigen divided by cpm in cultures without antigen was more than 2.

b. Lymphokine Production and Assay

T-cell clones (2×10⁵ cells/ml) were distributed to wells of 24-wellCostar plates with adherent cells from 1×10⁶ irradiated autologous PBMCplus antigen at optimal concentrations Cell free supernatants werecollected after 16 or 48 hours of incubation and stored at minus 20° C.until assayed for lymphokine activities IL-2 activity in supernatantsharvested after 16 hours of incubation was determined by their abilityto stimulate an IL-2-dependent mouse T-cell clone (CTLL 2) toproliferate [Mustafa et al., (1985) Clin. Exp. Immunol., 62:474-481].Granulocyte macrophage-CSF (GM-CSF) activity in the same supernatantswas assayed by the capacity of the supernatants to induce colonyformation in mononuclear bone marrow cells [Dahl et al., (1972) ActaPathol Microbiol Scand. Sect. B, 80:863-870]. Supernatants harvestedafter 48 hours were used to determine interferon-gamma activity by themethod of Dahl and Degree [Acta Pathol Microbiol. Scand. Sect. B,80:863-870], using human embryonic lung fibroblasts and vesicularstomatitis virus as the challenge virus

c. Cytotoxicity assay

Adherent cells from 1×10⁶ autologous irradiated PBMC in 24-well Costarplates were pulsed with antigens at optimal concentrations and thedensity of T cell clones was adjusted to 1×10⁵ cells/well After 7 daysof incubation, T cells were washed off, and 0.5 ml of 0.03% neutral red(in saline+10% FBS) were added to each well and the plates incubated for30 minutes Neutral red was then removed from the wells by washing, andthe dye taken up by macrophages was released by adding 0.5 ml of 0.05 Macetic acid in 50% ethanol [Parish et al., (1983) J. Immunol Methods,58:225]. Percentage cytotoxicity was calculated from spectrophotometricabsorbance measurement at 540 nanometers [0D₅₄₀ ] according to theformula: ##EQU1## where OD₅₄₀ con.=OD₅₄₀ of control cultures withadherent cells+T-cell clones; and OD540 study=OD540 of study cultureswith adherent cells+T-cell clones+antigen.

5. Peptide-Induced Pooled T Cell Stimulation

Stimulation assays of pooled human T cells were carried out for theinventors by Dr. Stefan Kaufman of the Max Plank Institute forImmunology, Freiberg, West Germany. Again, coded samples were suppliedfor the assays.

The assay procedure was as follows. Mononuclear cells were isolated fromperipheral blood of M. bovis BCG-vaccinated persons on Ficoll-Hypaquegradients [Emmerich et al., (1986) J. Exp. Med., 163:1024-1029; andBoyum, (1968) Scand. J. Clin. Lab. Invest., 21 (Suppl. 97):31], and wereused to seed wells of a 96-well microtiter plate at about 2×10⁵cells/well. Antigen was then added at 0.1 ug/ml, 1 ug/ml and 10 ug/ml.

After six days of culture, 1 microCurie (uCi) of [³ H]-thymidine wasadded to each well. Eighteen hours later, cultures were harvested onglass fiber filters. Thymidine incorporation was measured in a liquidscintillation counter.

For the assays of Table 5, the PBMC were pooled. For the studiesconducted related to HLA restrictions, the PBMC were kept separate andthe HLA alleles ascertained by standard techniques.

The present invention has been described with respect to preferredembodiments. It will be clear to those skilled in the art thatmodifications and/or variations of the disclosed subject matter can bemade without departing from the scope of the invention set forth herein.

What is claimed is:
 1. A polymer comprising a plurality of peptiderepeating units, each of said peptide repeating units consistingessentially of an amino acid residue sequence that correspondssubstantially to a sequence of a 65KD mycobacterial cell wall protein-a,said peptide repeating units having the capacity of stimulating T cellsimmune to the mycobacteria of said 65KD mycobacterial cell wallprotein-a, wherein said peptide repeating units consist essentially of asequence, written from left to right and in the direction fromaminoterminus to carboxy-terminus, represented by a formula selectedfrom the group consisting ofA V L E D P Y I L L V S S K V; L L V S S K VS T V K D L L P; L L P L L E K V I G A G K P L; A I L T G G O V I S E EV G L; I A F N S G L E P G V V A E K; A R R G L E R G L N A L A D A V KV; E K I G A E L V K E V A K K; G L K R G I E K A V E K V T E T L; and IE D A V R N A K A A V E E G.
 2. The polymer of claim 1 wherein saidmycobacterium is M. tuberculosis or M. bovis.
 3. The polymer of claim 1wherein said peptide repeating units are bonded together by oxidizedcysteine residues present at the termini of said repeating units.
 4. Thepolymer of claim 1 wherein said mycobacterium is M. leprae, and saidpeptide repeating units consist essentially of a sequence, written fromleft to right and in the direction from amino-terminus tocarboxy-terminus, represented by a formula selected rom the groupconsisting ofA V L E E P Y I L L V S S K V; and I A F N S G M E P G V VE K.
 5. A polypeptide that simulates proliferation of M. leprae-immune Tcells, said polypeptide having an amino acid residue sequence selectedfrom the group consisting ofA V L E D P Y I L L V S S K V; L L V S S K VS T V K D L L P; L L P L L E K V I G A G K P L; A I L T G G Q V I S E EV G L; I A F N S G L E P G V V A E K; A R R G L E R G L N A L A D A V KV; E K I G A E L V K E V A K K; G L K R G I E K A V E K V T E T L; and IE D A V R N A K A A V E E G.
 6. The polypeptide of claim 5 whosesequence isA V L E D P Y I L L V S S K V.
 7. The polypeptide of claim 5whose sequence isL L V S S K V S T V K D L L P.
 8. The polypeptide ofclaim 5 whose sequence isL L P L L E K V I G A G K P L.
 9. Thepolypeptide of claim 5 whose sequence isA I L T G G Q V I S E E V G L.10. The polypeptide of claim 5 whose sequence isI A F N S G L E P G V VA E K.
 11. The polypeptide of claim 5 whose sequence isA R R G L E R G LN A L A D A V K V.
 12. The polypeptide of claim 5 whose sequence isE K IG A E L V K E V A K K.
 13. The polypeptide of claim 5 whose sequence isGL K R G I E K A V E K V T E T L.
 14. The polypeptide of claim 5 whosesequence isI E D A V R N A K A A V E E G.